Protein C production in non-human transgenic mammals

ABSTRACT

Methods for producing protein C in transgenic non-human mammals are disclosed. The protein C is modified at the two-chain cleavage site between the light and heavy chains of protein C from Lys-Arg to R 1  -R 2  -R 3  -R 4  where R 1  through R 4  are individually Arg or Lys. DNA segments encoding modified protein C are introduced into the germ line of a non-human mammals, and the mammal or its female progeny produces milk containing protein C expressed from the introduced DNA segments. The protein C expressed from the introduced DNA segments has anticoagulant activity when activated. Non-human mammalian embryos and transgenic non-human mammals carrying DNA segments encoding heterologous protein C are also disclosed.

This application corresponds to U.S. patent application Ser. No. 08/565,074, filed Nov. 30, 1995, which was converted to U.S. Provisional Application No. 60/019,692, filed Jun. 13, 1996, and this application claims benefit of U.S. Provisional Application 60/044,453filed Nov. 30, 1995.

BACKGROUND OF THE INVENTION

Protein C in its activated form plays an important role in regulating blood coagulation. The activated protein C, a serine protease, inactivates coagulation Factors Va and VIIIa by limited proteolysis. The coagulation cascade initiated by tissue injury, for example, is prevented from proceeding in an unimpeded chain-reaction beyond the area of injury by activated protein C.

Protein C is synthesized in the liver as a single chain precursor polypeptide which is subsequently processed to a light chain of about 155 amino acids (M_(r) =21,000) and a heavy chain of 262 amino acids (M_(r) =40,000). The heavy and light chains circulate in the blood as a two-chain inactive protein, or zymogen, held together by a disulfide bond. When a 12 amino acid residue peptide is cleaved from the amino terminus of the heavy chain portion of the zymogen in a reaction mediated by thrombin, the protein becomes activated. The N-terminal portion of the light chain contains nine γ-carboxyglutamic acid (Gla) residues that are required for the calcium-dependent membrane binding and activation of the molecule. Another blood protein, referred to as "protein S", is believed to accelerate the protein C-catalyzed proteolysis of Factor Va.

Protein C has also been implicated in the action of tissue-type plasminogen activator (Kisiel et al., Behring Inst. Mitt. 73:29-42, 1983). Infusion of bovine activated protein C (APC) into dogs results in increased plasminogen activator activity (Comp et al., J. Clin. Invest. 68:1221-1228, 1981). Other studies (Sakata et al., Proc. Natl. Acad. Sci. USA 82:1121-1125, 1985) have shown that addition of APC to cultured endothelial cells leads to a rapid, dose-dependent increase in fibrinolytic activity in the conditioned media, reflecting increases in the activity of both urokinase-related and tissue-type plasminogen activators. APC treatment also results in a dose-dependent decrease in anti-activator activity. In addition, studies with monoclonal antibodies against endogenous APC (Snow et al., FASEB Abstracts, 1988) implicate APC in maintaining patency of arteries during fibrinolysis and limiting the extent of tissue infarct.

Experimental evidence indicates that protein C may be clinically useful in the treatment of thrombosis. Several studies with baboon models of thrombosis have indicated that activated protein C in low doses will be effective in prevention of fibrin deposition, platelet deposition and loss of circulation (Gruber et al., Hemostasis and Thrombosis 374a: abstract 1512, 1988; Widrow et al., Fibrinolysis 2 suppl. 1: abstract 7, 1988; Griffin et al., Thromb. Haemostasis 62: abstract 1512, 1989).

In addition, exogenous activated protein C has been shown to prevent the coagulopathic and lethal effects of gram negative septicemia (Taylor et al., J. Clin. Invest. 19:918-925, 1987). Data obtained from studies with baboons suggest that activated protein C plays a natural role in protecting against septicemia.

Until recently, protein C was purified from clotting factor concentrates (Marlar et al., Blood 59:1067-1072, 1982) or from plasma (Kisiel, J. Clin. Invest. 64:761-769, 1979) and activated in vitro. However, the possibility that the resulting product could be contaminated with such infectious agents as hepatitis virus, cytomegalovirus, or human immunodeficiency virus (HIV) make the process unfavorable.

While expression of protein C through recombinant means has been theoretically possible as the genes for both human and bovine protein C are known (Foster et al., Proc. Natl. Acad. Sci. USA 82:4673-4677, 1985; Foster et al., Proc. Natl. Acad Sci. USA 81:4766-4770, 1984 and U.S. Pat. No. 4,775,624), it has been met with limited success. Expression of some vitamin K-dependent proteins, such as protein C in cultured cells, has not produced protein C that has been at both commercially valuable levels and biologically functional when activated (i.e. had anticoagulant activity (Grinnell et al., in Bruley and Drohn, eds., Protein C and Related Anticoagulants:29-63, Gulf Publishing, Houston, Tex. and Grinnell et al., Bio/Technol. 5:1189-1192, 1987)). Transgenic expression of protein C has yielded somewhat higher levels of expression, but the recombinant protein's anticoagulant activity has still remained low, with less than 50% of the material having biological activity (Velander et al., Proc. Natl. Acad. Sci. USA 89:12003-12007, 1992). Therefore, there remains a need for producing protein C that is both expressed at high levels and has therapeutic value.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide methods for producing protein C in transgenic animals. It is a further object to provide transgenic animals that express human protein C in a mammary gland.

Within one aspect, the present invention provides methods for producing protein C in a transgenic animal comprising (a) providing a DNA construct comprising a first DNA segment encoding a secretion signal and a protein C propeptide operably linked to a second DNA segment encoding protein C, wherein the encoded protein C comprises a two-chain cleavage site modified from Lys-Arg to R₁ -R₂ -R₃ -R₄, and wherein each of R₁ -R₄ is individually Lys or Arg, and wherein said first and second segments are operably linked to additional DNA segments required for expression of the protein C DNA in a lactating mammary gland of a host female animal; (b) introducing said DNA construct into a fertilized egg of a non-human mammalian species; (c) inserting said egg into an oviduct or uterus of a female of said species to obtain offspring carrying said DNA construct; (d) breeding said offspring to produce female progeny that express said first and second DNA segments and produce milk containing protein C encoded by said second segment, wherein said protein has anticoagulant activity upon activation; (e) collecting milk from said female progeny; and (f) recovering the protein C from the milk. In one embodiment, R₁ -R₂ -R₃ -R₄ is Arg-Arg-Lys-Arg (SEQ ID NO: 20). In another embodiment, the method further comprises the step of activating the protein C. In another embodiment, the non-human mammalian species is selected from sheep, rabbits, cattle and goats. In another embodiment each of the first and second DNA segments comprises an intron. In another embodiment, the second DNA segment comprises a DNA sequence of nucleotides as shown in SEQ ID NO: 1 or SEQ ID NO:3. In another embodiment, the additional DNA segments comprise a transcriptional promoter selected from the group consisting of casein, β-lactoglobulin, α-lactoglobulin, α-lactalbumin and whey acidic protein gene promoters.

In another aspect, the present invention provides a transgenic non-human female mammal that produces recoverable amounts of human protein C in its milk, wherein at least 90% of the human protein C in the milk is two-chain protein C.

In another aspect, the present invention provides a process for producing a transgenic offspring of a mammal comprising the steps of (a) providing a DNA construct comprising a first DNA segment encoding a secretion signal and a protein C propeptide operably linked to a second DNA segment encoding protein C, wherein the encoded protein C comprises a two-chain cleavage site modified from Lys-Arg to R₁ -R₂ -R₃ -R₄, and wherein each of R₁ -R₄ is individually Lys or Arg, and wherein said first and second segments are operably linked to additional DNA segments required for expression of the protein C DNA in a lactating mammary gland of a host female animal; (b) introducing said DNA construct into a fertilized egg of a non-human mammalian species; and (c) inserting said egg into an oviduct or uterus of a female of said species to obtain offspring carrying said DNA construct.

Within another aspect, the present invention provides non-human mammals produced according to the process for producing a transgenic offspring of a mammal comprising the steps of (a) providing a DNA construct comprising a first DNA segment encoding a secretion signal and a protein C propeptide operably linked to a second DNA segment encoding protein C, wherein the encoded protein C comprises a two-chain cleavage site modified from Lys-Arg to R₁ -R₂ -R₃ -R₄, and wherein each of R₁ -R₄ is individually Lys or Arg, and wherein said first and second segments are operably linked to additional DNA segments required for expression of the protein C DNA in a lactating mammary gland of a host female animal; (b) introducing said DNA construct into a fertilized egg of a non-human mammalian species; and (c) inserting said egg into an oviduct or uterus of a female of said species to obtain offspring carrying said DNA construct.

In another aspect, the present invention provides a non-human mammalian embryo containing in its nucleus a heterologous DNA segment encoding protein C, wherein the encoded protein C comprises a two-chain cleavage site modified from Lys-Arg to R₁ -R₂ -R₃ -R₄, and wherein each of R₁ -R₄ is individually Lys or Arg.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates analysis of plasma-derived and transgenic protein C run under non-reducing and reducing conditions. Lane 1 is plasma-derived protein C and lane 2 is transgenic protein C from the milk of sheep 30851.

FIG. 2 illustrates sequencing of protein C from sheep line 30851. The initial yields were prosequence=9 pmol, light chain=563 pmol and heavy chain=565 pmol.

FIG. 3 illustrates clotting activity of transgenic protein C compared to plasma-derived protein C.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it will be helpful to define certain terms used herein:

As used herein, the term "biologically active" is used to denote protein C that is characterized by its anticoagulant and fibrinolytic properties. Protein C, when activated, inactivates factor Va and factor VIIIa in the presence of phospholipid and calcium. Activated protein C also enhances fibrinolysis, an effect believed to be mediated by the lowering of the levels of plasminogen activator inhibitors. As stated previously, two-chain protein C is activated upon cleavage of a 12 amino acid peptide from the amino terminus of the heavy chain portion of the zymogen.

The term "egg" is used to denote an unfertilized ovum, a fertilized ovum prior to fusion of the pronuclei or an early stage embryo (fertilized ovum with fused pronuclei).

A "female mammal that produces milk containing biologically active protein C" is one that, following pregnancy and delivery, produces, during the lactation period, milk containing recoverable amounts of protein C that can be activated to be biologically active. Those skilled in the art will recognized that such animals will naturally produce milk, and therefore the protein C, discontinuously.

The term "progeny" is used in its usual sense to include offspring and descendants.

The term "heterologous" is used to denote genetic material originating from a different species than that into which it has been introduced, or a protein produced from such genetic material.

Within the present invention, transgenic animal technology is employed to produce protein C within a mammary gland of a host female mammal. Expression in the mammary gland and subsequent secretion of the protein of interest into the milk overcomes many difficulties encountered in isolating proteins from other sources. Milk is readily collected, available in large quantities, and well characterized biochemically. Furthermore, the major milk proteins are present in milk at high concentrations (from about 1 to 16 g/l).

From a commercial point of view, it is clearly preferable to use as the host a species that has a large milk yield. While smaller animals such as mice and rats can be used (and are preferred at the proof-of-concept stage), within the present invention it is preferred to use livestock mammals including sheep and cattle. Sheep are particularly preferred due to such factors as the previous history of transgenesis in this species, milk yield, generation time, cost and the ready availability of equipment for collecting sheep milk. It is generally desirable to select a breed of host animal that has been bred for dairy use, such as East Friesland sheep, or to introduce dairy stock by breeding of the transgenic line at a later date. In any event, animals of known, good health status should be used.

Cloned DNA sequences encoding human protein C have been described (Foster and Davie, Proc. Natl. Acad. Sci. USA 81:4766-4770, 1984; Foster et al., Proc. Natl. Acad. USA 82:4673-4677, 1985; and Bang et al., U.S. Pat. No. 4,755,624, each incorporated herein by reference). Complementary cDNAs encoding protein C can be obtained from libraries prepared from liver cells of various mammalian species according to standard laboratory procedures. DNAs from other species, such as the protein C encoded by rats, pigs, sheep, cows and primates can be used and can be identified using probes from human cDNA.

In a preferred embodiment, human genomic DNAs encoding protein C are used. The human protein C gene is composed of nine exons ranging in size from 25 to 885 nucleotides, and seven introns ranging in size from 92 to 2668 nucleotides (U.S. Pat. No. 4,959,318, incorporated herein by reference). The first exon is non-coding and referred to as exon O. Exon I and a portion of exon II code for the 42 amino acid signal sequence and propeptide (i.e., pre-propeptide). The remaining portion of exon II, exon III, exon IV, exon V and a portion of exon VI code for the light chain of protein C. The remaining portion of exon VI, exon VII and exon VIII code for the heavy chain of protein C. A representative human genomic DNA sequence and corresponding amino acid sequence are shown in SEQ ID NOS: 1 and 2, respectively. A representative human protein C cDNA sequence and corresponding amino acid sequences are shown in SEQ ID NO: 3 and 4, respectively.

Those skilled in the art will recognize that naturally occurring allelic variants of these sequences will exist; that additional variants can be generated by amino acid substitution, deletion, or insertion; and that such variants are useful within the present invention. In general, it is preferred that any engineered variants comprise only a limited number of amino acid substitutions, deletions, or insertions, and that any substitutions are conservative. Thus, it is preferred to produce protein C polypeptides that are at least 90%, and more preferably at least 95% or more identical in sequence to the corresponding native protein.

Within the present invention, the proteolytic processing involved in the maturation of recombinant protein C from single chain form to the two-chain form (i.e., cleaved between the light chain and the heavy chain) has been enhanced by modifying the amino acid sequence around the two-chain cleavage site. In the normal situation, endoproteolytic cleavage of the precursor molecule at the Arg₁₅₇ -Asp₁₅₈ bond and the removal of the dipeptide Lys₁₅₆ -Arg₁₅₇ by a carboxypeptidase activity generate the light and heavy chains of protein C prior to secretion. Expression of protein C with the native (Lys-Arg) two-chain cleavage site produces protein C that may contain up to 40% or more uncleaved, single-chain protein C (Grinnel et al., in Protein C and Related Anticoagulants, eds., Bruley and Drohan, Gulf, Houston, pp. 29-63, 1990; Suttie, Thromb. Res. 44:129-134, 1986 and Yan et al., Trends Biochem. Sci. 14:264-268, 1989). The single-chain form of protein C may not be able to be activated. The cleavage site may be in the form of the amino acid sequence R₁ -R₂ -R₃ -R₄, wherein each of R1 through R4 is individually lysine (Lys) or arginine (Arg). Particularly preferred sequences include Arg-Arg-Lys-Arg (SEQ ID NO: 20) and Lys-Arg-Lys-Arg (SEQ ID NO: 21).

In a preferred embodiment, the present invention provides for recoverable amounts of human protein C in the milk of a non-human mammal, where at least 90%, preferably at least 95%, of the human protein C is two-chain protein C.

To obtain expression in the mammary gland, a transcription promoter from a milk protein gene is used. Milk protein genes include those genes encoding caseins, beta-lactoglobulin (BLG), α-lactalbumin, and whey acidic protein. The beta-lactoglobulin promoter is preferred. In the case of the ovine beta-lactoglobulin gene, a region of at least the proximal 406 bp of 5' flanking sequence of the ovine BLG gene (contained within nucleotides 3844 to 4257 of SEQ ID NO: 5) will generally be used. Larger portions of the 5' flanking sequence, up to about 5 kb, are preferred. A larger DNA segment encompassing the 5' flanking promoter region and the region encoding the 5' non-coding portion of the beta-lactoglobulin gene (contained within nucleotides 1 to 4257 of SEQ ID NO: 5) is particularly preferred. See Whitelaw et al., Biochem J. 286: 31-39, 1992. Similar fragments of promoter DNA from other species are also suitable.

Other regions of the beta-lactoglobulin gene may also be incorporated in constructs, as may genomic regions of the gene to be expressed. It is generally accepted in the art that constructs lacking introns, for example, express poorly in the transgenic lactating mammary gland in comparison with those constructs that contain introns (see Brinster et al., Proc. Natl. Acad. Sci. USA 85: 836-840, 1988; Palmiter et al., Proc. Natl. Acad. Sci. USA 88: 478-482, 1991; Whitelaw et al., Transgenic Res. 1: 3-13, 1991; WO 89/01343; WO 91/02318). In this regard, it is generally preferred, where possible, to use genomic sequences containing all or some of the native introns of a gene encoding protein C. Within certain embodiments of the invention, the further inclusion of at least some introns from the beta-lactoglobulin gene is preferred. One such region is a DNA segment which provides for intron splicing and RNA polyadenylation from the 3' non-coding region of the ovine beta-lactoglobulin gene. When substituted for the natural 3' non-coding sequences of a gene, this ovine beta-lactoglobulin segment can both enhance and stabilize expression levels of the protein C.

For expression of protein C, DNA segments encoding protein C are operably linked to additional DNA segments required for their expression to produce expression units. One such additional segment is the above-mentioned milk protein gene promoter. Sequences allowing for termination of transcription and polyadenylation of mRNA may also be incorporated. Such sequences are well known in the art, for example, one such termination sequence is the "upstream mouse sequence" (McGeady et al., DNA 5:289-298,1986). The expression units will further include a DNA segment encoding a secretion signal operably linked to the segment encoding the protein C polypeptide chain The secretion signal may be a native protein C secretion signal or may be that of another protein, such as a milk protein. The term "secretion signal" is used herein to denote that portion of a protein that directs it through the secretory pathway of a cell to the outside. Secretion signals are most commonly found at the amino termini of proteins. See, for example, von Heinje, Nuc. Acids Res. 14: 4683-4690, 1986; and Meade et al., U.S. Pat. No. 4,873,316, which are incorporated herein by reference.

Construction of expression units is conveniently carried out by inserting a protein C sequence into a plasmid or phage vector containing the additional DNA segments, although the expression unit may be constructed by essentially any sequence of ligations. It is particularly convenient to provide a vector containing a DNA segment encoding a milk protein and to replace the coding sequence for the milk protein with that of a protein C (including a secretion signal), thereby creating a gene fusion that includes the expression control sequences of the milk protein gene. In any event, cloning of the expression units in plasmids or other vectors facilitates the amplification of the protein C sequences. Amplification is conveniently carried out in bacterial (e.g. E. coli) host cells, thus the vectors will typically include an origin of replication and a selectable marker functional in bacterial host cells.

The expression unit is then introduced into fertilized eggs (including early-stage embryos) of the chosen host species. Introduction of heterologous DNA can be accomplished by one of several routes, including pronuclear microinjection (e.g. U.S. Pat. No. 4,873,191), retroviral infection (Jaenisch, Science 240: 1468-1474, 1988) or site-directed integration using embryonic stem (ES) cells (reviewed by Bradley et al., Bio/Technology 10: 534-539, 1992). The eggs are then implanted into the oviducts or uteri of pseudopregnant females and allowed to develop to term. Offspring carrying the introduced DNA in their germ line can pass the DNA on to their progeny in the normal, Mendelian fashion, allowing the development of transgenic herds. General procedures for producing transgenic animals are known in the art. See, for example, Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1986; Simons et al., Bio/Technology 6: 179-183, 1988; Wall et al., Biol. Reprod. 32: 645-651, 1985; Buhler et al., Bio/Technology 8: 140-143, 1990; Ebert et al., Bio/Technology 9: 835-838, 1991; Krimpenfort et al., Bio/Technology 9: 844-847, 1991; Wall et al., J. Cell, Biochem. 49: 113-120, 1992; and WIPO publications WO 88/00239, WO 90/05188, WO 92/11757; and GB 87/00458, which are incorporated herein by reference. Techniques for introducing foreign DNA sequences into mammals and their germ cells were originally developed in the mouse. See, e.g., Gordon et al., Proc. Natl. Acad. Sci. USA 77: 7380-7384, 1980; Gordon and Ruddle, Science 214: 1244-1246, 1981; Palmiter and Brinster, Cell 41: 343-345, 1985; Brinster et al., Proc. Natl. Acad. Sci. USA 82: 4438-4442, 1985; and Hogan et al. (ibid.). These techniques were subsequently adapted for use with larger animals, including livestock species (see e.g., WIPO publications WO 88/00239, WO 90/05188, and WO 92/11757; and Simons et al., Bio/Technology 6: 179-183, 1988). To summarize, in the most efficient route used to date in the generation of transgenic mice or livestock, several hundred linear molecules of the DNA of interest are injected into one of the pro-nuclei of a fertilized egg. Injection of DNA into the cytoplasm of a zygote can also be employed.

In general, female animals are superovulated by treatment with follicle stimulating hormone, then mated. Fertilized eggs are collected, and the heterologous DNA is injected into the eggs using known methods. See, for example, U.S. Pat. No. 4,873,191; Gordon et al., Proc. Natl. Acad. Sci. USA 82: 7380-7384, 1980; Gordon and Ruddle, Science 214: 1244-1246, 1981; Palmiter and Brinster, Cell 41: 343-345, 1985; Brinster et al., Proc. Natl. Acad, Sci. USA 82: 4438-4442, 1985; Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1986; Simons et al. Bio/Technology 6: 179-183, 1988; Wall et al., Biol. Reprod. 32: 645-651, 1985; Buhler et al., Bio/Technology 8: 140-143, 1990; Ebert et al., Bio/Technology 9: 835-838, 1991; Krimpenfort et al., Bio/Technology 9: 844-847, 1991; Wall et al., J. Cell. Biochem. 49: 113-120, 1992; WIPO publications WO 88/00239, WO 90/05118, and WO 92/11757; and GB 87/00458, which are incorporated herein by reference.

For injection into fertilized eggs, the expression units are removed from their respective vectors by digestion with appropriate restriction enzymes. For convenience, it is preferred to design the vectors so that the expression units are removed by cleavage with enzymes that do not cut either within the expression units or elsewhere in the vectors. The expression units are recovered by conventional methods, such as electro-elution followed by phenol extraction and ethanol precipitation, sucrose density gradient centrifugation, or combinations of these approaches.

DNA is injected into eggs essentially as described in Hogan et al., ibid. In a typical injection, eggs in a dish of an embryo culture medium are located using a stereo zoom microscope (×50 or ×63 magnification preferred). Suitable media include Hepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid) or bicarbonate buffered media such as M2 or M16 (available from Sigma Chemical Co., St. Louis, USA) or synthetic oviduct medium (disclosed below). The eggs are secured and transferred to the center of a glass slide on an injection rig using, for example, a drummond pipette complete with capillary tube. Viewing at lower (e.g. ×4) magnification is used at this stage. Using the holding pipette of the injection rig, the eggs are positioned centrally on the slide. Individual eggs are sequentially secured to the holding pipette for injection. For each injection process, the holding pipette/egg is positioned in the center of the viewing field. The injection needle is then positioned directly below the egg. Preferably using ×40 Nomarski objectives, both manipulator heights are adjusted to focus both the egg and the needle. The pronuclei are located by rotating the egg and adjusting the holding pipette assembly as necessary. Once the pronucleus has been located, the height of the manipulator is altered to focus the pronuclear membrane. The injection needle is positioned below the egg such that the needle tip is in a position below the center of the pronucleus. The position of the needle is then altered using the injection manipulator assembly to bring the needle and the pronucleus into the same focal plane. The needle is moved, via the joy stick on the injection manipulator assembly, to a position to the right of the egg. With a short, continuous jabbing movement, the pronuclear membrane is pierced to leave the needle tip inside the pronucleus. Pressure is applied to the injection needle via, for example, a glass syringe until the pronucleus swells to approximately twice its volume. At this point, the needle is slowly removed. Reverting to lower (e.g. ×4) magnification, the injected egg is moved to a different area of the slide, and the process is repeated with another egg.

After the DNA is injected, the eggs may be cultured to allow the pronuclei to fuse, producing one-cell or later stage embryos. In general, the eggs are cultured at approximately the body temperature of the species used in a buffered medium containing balanced salts and serum. Surviving embryos are then transferred to pseudopregnant recipient females, typically by inserting them into the oviduct or uterus, and allowed to develop to term. During embryogenesis, some of the injected DNA integrates in a random fashion in the genomes of a small number of the developing embryos.

Potential transgenic offspring are screened via blood samples and/or tissue biopsies. DNA is prepared from these samples and examined for the presence of the injected construct by techniques such as polymerase chain reaction (PCR; see Mullis, U.S. Pat. No. 4,683,202) and Southern blotting (Southern, J. Mol. Biol. 98:503, 1975; Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982). Founder transgenic animals, or GOs, may be wholly transgenic, having transgenes in all of their cells, or mosaic, having transgenes in only a subset of cells (see, for example, Wilkie et al., Develop. Biol. 118: 9-18, 1986). In the latter case, groups of germ cells may be wholly or partially transgenic. In the latter case, the number of transgenic progeny from a founder animal will be less than the expected 50% predicted from Mendelian principles. Founder G0 animals are grown to sexual maturity and mated to obtain offspring, or G1s. The G1s are also examined for the presence of the transgene to demonstrate transmission from founder G0 animals. In the case of male G0s, these may be mated with several non-transgenic females to generate many offspring. This increases the chances of observing transgene transmission. Female G0 founders may be mated naturally, artificially inseminated or superovulated to obtain many eggs which are transferred to surrogate mothers. The latter course gives the best chance of observing transmission in animals having a limited number of young. The above-described breeding procedures are used to obtain animals that can pass the DNA on to subsequent generations of offspring in the normal, Mendelian fashion, allowing the development of, for example, colonies (mice), flocks (sheep), or herds (pigs, goats and cattle) of transgenic animals.

The milk from lactating G0 and G1 females is examined for the expression of the heterologous protein using immunological techniques such as ELISA (see Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988) and Western blotting (Towbin et al., Proc. Natl. Acad. Sci. USA 76: 4350-4354, 1979). For a variety of reasons known in the art, expression levels of the heterologous protein will be expected to differ between individuals.

A satisfactory family of animals should satisfy three criteria: they should be derived from the same founder G0 animal; they should exhibit stable transmission of the transgene; and they should exhibit acceptably stable expression levels from generation to generation and from lactation to lactation of individual animals. These principles have been demonstrated and discussed (Carver et al., Bio/Technology 11: 1263-1270, 1993). Animals from such a suitable family are referred to as a "line." Initially, male animals, G0 or G1, are used to derive a flock or herd of producer animals by natural or artificial insemination. In this way, many female animals containing the same transgene integration event can be quickly generated from which a supply of milk can be obtained.

The protein C is recovered from milk using standard practices such as skimming, precipitation, filtration and protein chromatography techniques.

Protein C produced according to the present invention can be activated by removal of the activation peptide from the amino terminus of the heavy chain. Activation can be achieved using methods that are well known in the art, for example, using α-thrombin (Marlar et al., Blood 59:1067-1072, 1982), trypsin (Marlar et al., 1982, ibid.), Russel's viper venom factor X activator (Kisiel, J. Clin. Invest. 64:761-769, 1979) or commercially available Protac C (American Diagnostica, NY, N.Y.).

The protein C molecules provided by the present invention and pharmaceutical compositions thereof are particularly useful for administration to humans to treat a variety of conditions involving intravascular coagulation. For instance, although deep vein thrombosis and pulmonary embolism can be treated with conventional anticoagulants, the activated protein C described herein may be used to prevent the occurrence of thromboembolic complications in identified high risk patients, such as those undergoing surgery or those with congestive heart failure. Since activated protein C is more selective than heparin, being active in the body generally when and where thrombin is generated and fibrin thrombi are formed, activated protein C will be more effective and less likely to cause bleeding complications than heparin when used prophylactically for the prevention of deep vein thrombosis. The dose of activated protein C for prevention of deep vein thrombosis is in the range of about 100 μg to 100 mg/day, and administration should begin at least about 6 hours prior to surgery and continue at least until the patient becomes ambulatory. In established deep vein thrombosis and/or pulmonary embolism, the dose of activated protein C ranges from about 100 μg to 100 mg as a loading dose followed by maintenance doses ranging from 3 to 300 mg/day. Because of the lower likelihood of bleeding complications from activated protein C infusions, activated protein C can replace or lower the dose of heparin during or after surgery in conjunction with thrombectomies or embolectomies.

The activated protein C compositions of the present invention will also have substantial utility in the prevention of cardiogenic emboli and in the treatment of thrombotic strokes. Because of its low potential for causing bleeding complications and its selectivity, activated protein C can be given to stroke victims and may prevent the extension of the occluding arterial thrombus. The amount of activated protein C administered will vary with each patient depending on the nature and severity of the stroke, but doses will generally be in the range of those suggested below.

Pharmaceutical compositions of activated protein C provided herein will be a useful treatment in acute myocardial infarction because of the ability of activated protein C to enhance in vitro fibrinolysis. Activated protein C can be given with tissue plasminogen activator or streptokinase during the acute phases of the myocardial infarction. After the occluding coronary thrombus is dissolved, activated protein C can be given for subsequent days or weeks to prevent coronary reocculsion. In acute myocardial infarction, the patient is given a loading dose of at least about 1-500 mg of activated protein C, followed by maintenance doses of 1-100 mg/day.

Activated protein C is useful in the treatment of disseminated intravascular coagulation (DIC). Patients with DIC characteristically have widespread microcirculatory thrombi and often severe bleeding problems which result from consumption of essential clotting factors. Because of its selectivity, activated protein C will not aggravate the bleeding problems associated with DIC, as do conventional anticoagulants, but will retard or inhibit the formation of additional microvascular fibrin deposits.

The invention is further illustrated by the following non-limiting examples.

EXAMPLES Example I

A. Vector pMAD6 Construction

The multiple cloning site of the vector pUC18 (Yanisch-Perron et al., Gene 33:103-119, 1985) was removed and replaced with a synthetic double stranded oligonucleotide (the strands of which are shown in SEQ ID NO: 6 and SEQ ID NO: 7) containing the restriction sites Pvu I/Mlu I/Eco RV/Xba I/Pvu I/Mlu I, and flanked by 5' overhangs compatible with the restriction sites Eco RI and Hind III. pUC18 was cleaved with both Eco RI and Hind III, the 5' terminal phosphate groups were removed with calf intestinal phosphatase, and the oligonucleotide was ligated into the vector backbone. The DNA sequence across the junction was confirmed by sequencing, and the new plasmid was called pUCPM.

The β-lactoglobulin (BLG) gene sequences from pSS1tgXS (disclosed in WIPO publication WO 88/00239) were excised as a Sal I-Xba I fragment and recloned into the vector pUCPM that had been cut with Sal I and Xba I to construct vector pUCXS. pUCXS is thus a pUC18 derivative containing the entire BLG gene from the Sal I site to the Xba I site of phage SS1 (Ali and Clark, J. Mol. Biol. 199: 415-426, 1988).

The plasmid pSS1tgSE (disclosed in WIPO publication WO 88/00239) contains a 1290 bp BLG fragment flanked by Sph I and EcoR I restriction sites, a region spanning a unique Not I site and a single Pvu II site which lies in the 5' untranslated leader of the BLG mRNA. Into this Pvu II site was ligated a double stranded, 8 bp DNA linker (5'-GGATATCC-3') encoding the recognition site for the enzyme Eco RV. This plasmid was called pSS1tgSE/RV. DNA sequences bounded by Sph I and Not I restriction sites in pSS1tgSE/RV were excised by enzymatic digestion and used to replace the equivalent fragment in pUCXS. The resulting plasmid was called pUCXSRV. The sequence of the BLG insert in pUCXSRV is shown in SEQ ID NO: 5, with the unique Eco RV site at nucleotide 4245 in the 5' untranslated leader region of the BLG gene. This site allows insertion of any additional DNA sequences under the control of the BLG promoter 3' to the transcription initiation site.

Using the primers BLGAMP3 (5'-TGG ATC CCC TGC CGG TGC CTC TGG-3'; SEQ ID NO: 8) and BLGAMP4 (5'-AAC GCG TCA TCC TCT GTG AGC CAG-3'; SEQ ID NO: 9) a PCR fragment of approximately 650 bp was produced from sequences immediately 3' to the stop codon of the BLG gene in pUCXSRV. The PCR fragment was engineered to have a BamH I site at its 51 end and an Mlu I site at its 3' end and was cloned as such into BamH I and Mlu I cut pGEM7zf(+) (Promega) to give pDAM200(+).

pUCXSRV was digested with Kpn I, and the largest, vector containing band was gel purified. This band contained the entire pUC plasmid sequences and some 3' non-coding sequences from the BLG gene. Into this backbone was ligated the small Kpn I fragment from pDAM200(+) which, in the correct orientation, effectively engineered a Bam HI site at the extreme 5' end of the 2.6 Kbp of the BLG 3' flanking region. This plasmid was called pBLAC200. A 2.6 Kbp Cla I-Xba I fragment from pBLAC200 was ligated into Cla I-Xba I cut pSP72 vector (Promega), thus placing an Eco RV site immediately upstream of the BLG sequences. This plasmid was called pBLAC210.

The 2.6 Kbp Eco RV-Xba I fragment from pBLAC210 was ligated into Eco RV-Xba I cut pUCXSRV to form pMAD6 (SEQ ID NO: 23). This, in effect, excised all coding and intron sequences from pUCXSRV, forming a BLG minigene consisting of 4.2 Kbp of 5' promoter and 2.6 Kbp of 3' downstream sequences flanking a unique Eco RV site. An oligonucleotide linker (ZC6839: ACTACGTAGT; SEQ ID NO: 10) was inserted into the Eco RV site of pMAD6 (SEQ ID NO: 23). This modification destroyed the Eco RV site and created a Sna BI site to be used for cloning purposes. The vector was designated pMAD6-Sna. Messenger RNA initiates upstream of the Sna BI site and terminates downstream of the Sna BI site. The precursor transcript will encode a single BLG-derived intron, intron 6, which is entirely within the 3' untranslated region of the gene.

B. Intronless Vector pMAD

The beta-lactoglobulin cloning vector pMAD was also constructed to allow the insertion of cDNAs under the control of the beta-lactoglobulin gene promoter in constructs containing no introns. To generate pMAD, the plasmid pBLAC100 was opened by digestion with both Eco RV and Sal I. The vector fragment was gel purified and the linearized vector was ligated with the 4.2 kb promoter fragment from the plasmid pUCXSRV as a Sal I-Eco RV fragment. The resulting construct was designated pST1 and constitutes a beta-lactoglobulin mini-gene encompassing a 4.2 kb of promoter region and 2.1 kb of 3' non-coding region beginning immediately downstream of the beta-lactoglobuling translational termination codon. A unique Eco RV site allows blunt-end cloning of any additional DNA sequences. To generate transgenic animals it is generally accepted in the art and preferred to separate bacterial plasmid vector sequences from those intended to be used in the generation of transgenic animals. In order to allow the practical excision of novel cDNA based constructs using this beta-lactoglobulin mini-gene, the minigene was excised from pST1 on a Xho I-Not I fragment, the DNA termini made flush with Klenow polymerase and the product was ligated into the Eco RV site of pUCPM to yield pMAD. Digestion with Mlu I liberates beta-lactoglobulin-cDNA constructs from the bacterial vector backbone.

Intronless constructs based on cDNAs and vectors such as pMAD benefit from the use of "rescue technology" for efficient expression. Rescue technology takes advantage of the ability of a co-injected and co-integrated BLG gene to improve the expression levels obtained from intronless, cDNA-based constructs in the transgenic system. Rescue technology is disclosed in WIPO publication WO 92/11358, and is incorporated herein by reference.

Example 2

A. Isolation of cDNA

A cDNA sequence coding for human protein C was prepared as described in U.S. Pat. No. 4,959,318, which is incorporated herein by reference. Briefly, a genomic fragment containing an exon corresponding to amino acids -42 to -19 (SEQ ID NO: 1) of the pre-pro peptide of protein C was isolated, nick translated and used as a probe to screen a cDNA library constructed by the technique of Gubler and Hoffman, Gene 25:263-269, 1983, using mRNA from HepG2 cells. This cell line was derived from human hepatocytes and was previously shown to synthesize protein C (Fair and Bahnak, Blood 64:194-204, 1984). Positive clones comprising cDNA inserted into the Eco RI site of phage λgtll were isolated and screened with an oligonucleotide probe corresponding to the 5' non-coding region of the protein C gene. One clone was also positive with this probe and its entire nucleotide sequence was determined. The cDNA contained 70 bp of 5' untranslated sequence, the entire coding sequence for human prepro-protein C, and the entire 3' non-coding region corresponding to the second polyadenylation site.

B. Subcloning of Protein C cDNA

The vector pDX was derived from pD3, which was generated from plasmid pDHFRIII (Berkner et al., Nuc. Acids Res. 13:841-857, 1985). The Pst I site immediately upstream from the DHFR sequence in pDHFRIII was converted to a Bcl I site by digestion with Pst I. The DNA was phenol extracted, ethanol precipitated and resuspended in buffer B (50 mM Tris pH 8, 7 mM MgCl₂, 7 mM β-MSH). A ligation reaction containing the linearized plasmid DNA and Bcl I linkers was done. The resulting plasmid was phenol extracted, ethanol precipitated and digested with Bcl I and gel purified. The gel purified plasmid DNA was circularized by ligation and used to transform E. coli HB101. Positive colonies were identified by restriction analysis and designated PDHFR'. DNA from positive colonies was isolated and used to transform dam⁻⁻ E. coli.

Plasmid pD2' was generated by cleaving pDHFR' and pSV40 (comprising Bam HI digested SV40 DNA cloned into the Bam HI site of pML-1 (Lusky et al., Nature 293:79-81, 1981)) with Bcl I and Bam HI. The DNA fragments were resolved by gel electrophoresis, and the 4.9 kb pDHFR' fragment and 0.2 kb SV40 fragment were isolated. These fragments were used in a ligation reaction, and the resulting plasmid, designated pD2', was used to transform E. coli RRI.

Plasmid pD2' was modified by deleting the "poison" sequences in the pBR322 region (Lusky et al., 1981, ibid.). Plasmids pD2' and pML-1 were digested with Eco RI and Nru I. The 1.7 kb pD2' fragment and 1.8 kb pML-1 fragment were isolated by gel purification, circularized in a ligation reaction and used to transform E. coli HB101. Positive colonies were identified using restriction analysis (designated pD2) and digested with Eco RI and Bcl I. A 2.8 kb fragment (fragment C) was isolated and gel purified.

To generate the remaining fragments used in constructing pD3, pDHFRIII was modified to convert the Sac II (Sst II) site into either a Hind III or Kpn I site. pDHFRIII was digested with Sst II and ligation reactions with either Hind III or Kpn I linkers were done. The resultant plasmids were digested with either Hind III or Kpn I and gel purified. The resultant plasmids were designated either pDHFRIII (Hind III) or pDHFRIII (Kpn I). A 700 bp KpnI-Bgl II fragment (fragment A) was purified from pDHFRIII (Hind III).

The SV40 enhancer sequence was inserted into pDHFRIII (Hind III) by first digesting SV40 DNA with Hind III, and DNA from 5089 to 968 bp was isolated and purified. Plasmid pDHFRIII (Hind III) was phosphatased, and the SV40 DNA and linearized plasmid pDHFRIII (Hind III) were used in a ligation reaction. A 700 bp Eco RI-Kpn I fragment (fragment B) was isolated from the resulting plasmid.

For the final construction of pD3, fragments A (50 ng), B (50 ng) and C (10 ng) were combined in a ligation reaction and used to transform E. coli RRI. Positive colonies were isolated and plasmid DNA was prepared.

Plasmid pD3 was modified to accept the insertion of the protein C sequence by converting the Bcl I insertion site to an Eco RI site. First, the Eco RI site present in pD3 (the leftmost terminus in adenovirus 5 0-1) was converted to a Bam HI site via conventional linkering procedures. The resultant plasmid was transformed in E. coli HB101. Plasmid DNA was prepared, and positive clones were identified by restriction analysis.

pD3' is a vector identical to pD3 except that the SV40 polyadenylation signal (i.e., the SV40 Bam HI (2533 bp) to Bcl I (2770 bp) fragment) is in the late orientation. Thus, pD3' contains a Bam HI site as the site of gene insertion.

To generate pDX, the Eco RI site in pD3' was converted to a Bcl I site by Eco RI cleavage, incubation with SI nuclease and subsequent ligation with Bcl I linkers. DNA was prepared from a positively identified colony, and a 1.9 kb Xho I-Pst I fragment containing the altered restriction site was prepared via gel purification. In a second modification, Bcl I-cleaved pD3 was ligated with Eco RI-Bcl I adapters in order to generate an Eco RI site as the position for inserting a gene into the expression vector. Positive colonies were identified by restriction analysis. The resulting plasmid, designated pDX, has a unique Eco RI site for insertion of foreign genes

The protein C cDNA was inserted into pDX as an Eco RI fragment. Plasmids were screened by restriction analysis. A plasmid having the protein C insert in the correct orientation with respect to the promoter elements and plasmid DNA was designated pDX/PC. Because the cDNA insert in pDX/PC contains a ATG codon in the 5' non-coding region, deletion mutagenesis was performed on the cDNA. Deletion of the three base pairs was performed according to standard procedures or oligonucleotide-directed mutagenesis. The pDX-based vector containing the modified cDNA was designated p594.

C. Modification of the Protein C Processing Site

To enhance the processing of single-chain protein C to the two-chain form, two additional arginine residues were introduced immediately upstream of the Lys₁₅₆ -Arg₁₅₇ cleavage site of the precursor protein, resulting in a cleavage site consisting of four basic amino acids, Arg-Arg-Lys-Arg (SEQ ID NO: 20). The resultant mutant precursor of protein C was designated PC962. It contains the sequence Ser-His-Leu-Arg-Arg-Lys-Arg-Asp (SEQ ID NO: 22) at the cleavage site. Processing at the Arg-Asp bond results in a two-chain protein C molecule.

The mutant molecule was generated by altering the cloned cDNA by site-specific mutagenesis (essentially as described by Zoller and Smith, DNA 3:479-488, 1984, for the two-primer method) using the mutagenic oligonucleotide ZC962 (⁵ 'AGTCACCTGAGAAGAAAACGAGACA³ '; SEQ ID NO: 11). Plasmid p594 was digested with Sst I, and the approximately 87 bp fragment was cloned into M13mp11 and single-stranded template DNA was isolated. Following mutagenesis, a correct clone was identified by sequencing. Replicative form DNA was isolated, digested with Sst I, and the protein C fragment was inserted into Sst I-cut p594. Clones having the Sst I fragment inserted in the desired orientation were identified by restriction enzyme mapping. The resulting expression vector was designated pDX/PC962.

D. Intronless Protein C Construct

To facilitate the cloning of the protein C cDNA, PC962, into pMAD, the cDNA contained in pDX/PC962 was modified to incorporate Eco RV sites at the extremities of the protein C cDNA insert. A 769 bp Sst II-Pst I fragment encompassing the 3' end of PC962 was cloned between the Sst II and Pst I sites of pBluescript II SK® (Stratagene, La Jolla, Calif.). The fragment was excised with Sst II and Eco RV and purified. The 5' portion of PC962 was modified by PCR. The sense oligonucleotide primer for this reaction covered the 5' ATG region of the cDNA and provided an Eco RV site upstream of this in the product. The antisense oligonucleotide primer covered the Sst II site used to generate the Sst II-Eco RV fragment. The resulting PCR product was digested with Eco RV and Sst II and ligated with the Sst II-Eco RV 3' fragment and Eco RV digested pMAD. The resulting plasmid, designated pCORP9 effectively contained the PC962 cDNA flanked by Eco RV sites in an intronless fusion driven by the beta-lactoglobulin promoter.

E. Genomic Protein C DNA Construction

A genomic DNA construct containing exons I through VIII was made. See, U.S. Pat. No. 4,959,318, which is incorporated herein by reference, for disclosure of the exon structure of the protein C gene. This genomic construct, designated GPC10-1, changed the sequence 16 base pairs upstream of the ATG from the native protein C sequence to the beta-lactoglobulin sequence and introduced mutations in the propeptide cleavage site located in exon 2, and the two-chain cleavage site located in exon 6, as described below.

The construct was assembled using four fragments designated A, B, C and D and encompassed the protein C gene sequence from the ATG to a Bam HI site in exon VIII, immediately upstream of the stop codon. The fragments were generated from a human genomic library in λ Charon 4A phage that was screened with a radiolabeled cDNA probe for human protein C. The screening of the λ library produced three clones that together mapped the entire protein C gene (Foster et al., 1985, ibid.). These clones were designated PCλ1, PCλ6 and PCλ8.

Fragment A was a Not I to Eco RI fragment that contained exons I and II of the genomic sequence and was 1698 bp. A subclone of PCλ6 contained an Eco RI to Eco RI fragment and was designated pHCR4.4-1. Using pHCR4.4-1 as a template and oligonucleotides ZC6303 (SEQ ID NO: 12) and ZC6337 (SEQ ID NO: 13), a DNA fragment was generated by polymerase chain reaction (PCR). Oligonucleotide ZC6303 changed the sequence 16 base pairs 5' to the ATG sequence from the native protein C sequence to the equivalent sequence from the beta-lactoglobulin gene and introduced a Not I site. Oligonucleotide ZC6337 changed the propeptide cleavage site from Arg-Ile-Arg-Lys-Arg (SEQ ID NO: 24) to Gln-Arg-Arg-Lys-Arg (SEQ ID NO: 25). The resulting PCR-generated fragment was digested with Not I and Bss HII, and a 1402 base pair fragment was gel purified and designated A1. A second fragment was prepared using a λ gt11 clone of PCλ1 as a template with oligonucleotides ZC6306 (SEQ ID NO: 14) and ZC6338 (SEQ ID NO: 15) in a polymerase chain reaction. The resulting DNA fragment, designated A3, was digested with Bss HII and Eco RI and gel purified, resulting in a 296 base pair fragment. Fragments A1 and A3 were ligated into the Bluescript II KS® phagemid vector (Stratagene, La Jolla, Calif.). The resulting plasmid, designated GPC 2-2, was digested with Not I and Eco RI, gel purified and the Not I-Eco RI DNA fragment was designated Fragment A.

pCR 2-14 is a subclone that contains an Eco RI to Eco RI DNA fragment of PCλ8 (Foster et al., 1985, ibid.). The plasmid was digested with Eco RI and Sst I and gel purified. The resulting fragment was designated Fragment B.

Plasmid pCR 2-14 was used as template DNA with oligonucleotides ZC6373 (SEQ ID NO: 16) and ZC6305 (SEQ ID NO: 17), which introduced an Afl II site and the RRKR mutation of the native (KR) two-chain cleavage site, in a polymerase chain reaction. The resulting PCR-generated fragment was digested with Bgl II and Afl II and gel purified, resulting in a 1441 base pair fragment, designated E1. Fragment E1 was used in a ligation reaction with oligonucleotides ZC6302 (SEQ ID NO: 18) and ZC6304 (SEQ ID NO: 19). These oligonucleotides form Afl II and Sst II restriction sites when annealed and were ligated to the 3' end of fragment E1, resulting in a fragment with a 5' Bgl II site and a 3' Sst II site. This fragment was used in a ligation reaction with a Bam HI-Sst II digested Bluescript II KS® phagemid vector (Stratagene). The resulting plasmid was designated GPC 8-5 and digested with Sst I and Sst II, generating a 626 base pair fragment, designated Fragment C.

A fourth fragment was generated by digestion of a genomic subclone (pHCB7-1) of PCλ8. pHCB7-1 contained a Bgl II to Bgl II fragment that encompassed exons VI through VIII. pHCB7-1 was digested with Sst II and Bam HI and a 2702 base pair fragment was gel purified. The fragment was designated Fragment D.

A five-part ligation reaction was prepared using Not I and Bam HI digested and linearized Bluescript II KS® phagemid vector (Stratagene) with Fragment A (5' Not I to 3' Eco RI) that contained exons I and II, Fragment B (5' Eco RI to 3' Sst I) that contained exons III, IV and V, Fragment C (5' Sst I to 3' Sst II) that contained the 5' portion of exon VI and Fragment D (5' Sst II to 3' Bam HI) that contained the remaining 3' portion of exon VI and exons VII and VIII. The resulting DNA was 8950 base pairs and designated GPC 10-1.

GPC10-1 was originally generated with BLG sequences and a Not I site upstream of the ATG initiator codon and modifications to both cleavage sites. A clone, designated pPC12/BS, was generated to ensure that the 5' Not I site of GPC10-1 would not introduce secondary structure into mRNA molecules that could hinder translation. pPC12/BS was generated using PCR amplification of a 1 kb Not I-Sca I fragment that covered the 5' region of the protein C gene and contained the wild-type ATG codon environment. This introduced an Eco RV site immediately downstream of the Not I site, adjacent to the ATG codon, and a Bam HI site was incorporated 3' of the Sca I site to facilitate cloning. Following a Not I/Bam HI digestion, the PCR product was cloned into Not I-Bam HI digested Bluescript II KS® phagemid vector (Stratagene). The Not I-Eco RV-Sca I fragment present in pPC12/BS was excised, purified and ligated to GPC10-1, which had been linearized with Not I and partially digested with Sca I (the pUC ampillicin gene has an internal Sca I site). The resulting clone was designated GPC10-2 and possesses an Eco RV site immediately upstream of the ATG initiator codon.

GPC10-1 and GPC10-2 both terminated at the final Bam HI site in exon VIII of the protein C gene. To reconstitute the 56 bp of sequence, ending at the termination codon, two oligonucleotides were synthesized with flanking Bam HI (5') and Bgl II (3') restriction sites. Following annealing of the oligonucleotides, the product was cloned into Bam HI digested pBST+ to generate plasmid pPC3'. pBST+ is a derivative of pBS (Stratagene) with a new polylinker. The addition of the polylinker added Bgl II, Xho I, Nar I and Cla I restriction sites from the vector polylinker downstream of the destroyed Bgl II site of the oligonucleotide construct.

The Not I-Bam HI fragment of GPC10-1 was subcloned into Not I/Bam HI digested pPC3' to add 3' coding sequences of protein C, the TAG termination codon followed by Bgl II-Xho I-Nar I-Cla I. The 3' region of the protein C gene beginning with the Eco RV site in intron V was excised from this plasmid on an Eco RV-Cla I fragment.

The Eco RV-Eco RV fragment from GPC10-2, covering the 5' portion of the protein C gene, and the above Eco RI-Cla I fragment covering the 3' portion of the protein C gene were combined between the Eco RV and Cla I sites of pMAD6 (SEQ ID NO: 23) to generate pCORP13. This effectively placed a genomic portion of the protein C gene with modified propeptide and two-chain cleavage sites under the control of the beta-lactoglobulin promoter.

A further genomic construct was generated from pCORP13 that contained only the modified two-chain cleavage site. This was achieved using PCR amplification to modify two fragments which resulting in restoration of the coding capability of exon 2 from the mutant Gln-Arg-Arg-Lys-Arg (SEQ ID NO: 25) to the wild-type Arg-Ile-Arg-Lys-Arg (SEQ ID NO: 24). pCORP13 was used as template for these reactions. The first fragment was 1.3 kb, which encompassed the 5' end of the protein C gene up to the Bam HI site in exon 2. For this reason, the sense primer was designed to add a Hind III site 5' to the Eco RV site proximal to the ATG initiation codon. The antisense primer was designed to restore the wild-type sequences in exon 2, which included a restored Bam HI site. A second fragment of 0.2 kb from the Bam HI site in exon 2 to the Xho I site in intron 2, was also amplified. The two fragments were combined in pGEMII (Promega, Madison, Wis.) to generate pGEMPC1.5. A 7.5 kb Xho I fragment from pCORP 13 was ligated to Xho I digested pGEMPC1.5 to generate a complete protein C genomic sequence covering exons 1-8 with a wild-type propeptide cleavage site and a modified two-chain cleavage site. The plasmid was designated pGEMPC14. The sequence was excised from pGEMPC14 as a Hind III/Sal I fragment. The DNA termini were repaired using a Klenow reaction and the fragment was blunt-end ligated into Eco RV digested pMAD6 (SEQ ID NO: 23) to generate pCORP14.

Example 3

Mice for initial breeding stocks (C57BL6J, CBACA) were obtained from Harlan Olac Ltd. (Bicester, UK). These were mated in pairs to produce F1 hybrid cross (B6CBAF1) for recipient females, superovulated females, stud males and vasectomized males. All animals were kept on a 14 hour light/10 hour dark cycle and fed water and food (Special Diet Services RM3, Edinburgh, Scotland) ad libitum.

Transgenic mice were generated essentially as described in Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1986, which is incorporated herein by reference in its entirety. Female B6CBAF1 animals were superovulated at 4-5 weeks of age by an i.p. injection of pregnant mares' serum gonadotrophin (FOLLIGON, Vet-Drug, Falkirk, Scotland) (5 iu) followed by an i.p. injection of human chorionic gonadotrophin (CHORULON, Vet-Drug, Falkirk, Scotland) (5 iu) 45 hours later. They were then mated with a stud male overnight. Such females were next examined for copulation plugs. Those that had mated were sacrificed, and their eggs were collected for microinjection.

DNA was injected into the fertilized eggs as described in Hogan et al. (ibid.). Briefly, the vector containing the protein C expression unit was digested with Mlu I, and the expression unit was isolated by sucrose gradient centrifugation. All chemicals used were reagent grade (Sigma Chemical Co., St. Louis, Mo., U.S.A.), and all solutions were sterile and nuclease-free. Solutions of 20% and 40% sucrose in 1 M NaCl, 20 mM Tris pH 8.0, 5 mM EDTA were prepared using UHP water and filter sterilized. A 30% sucrose solution was prepared by mixing equal volumes of the 20% and 40% solutions. A gradient was prepared by layering 0.5 ml steps of the 40%, 30% and 20% sucrose solutions into a 2 ml polyallomer tube and allowed to stand for one hour. 100 μl of DNA solution (max. 8 μg DNA) was loaded onto the top of the gradient, and the gradient was centrifuged for 17-20 hours at 26,000 rpm, 15° C. in a Beckman TL100 ultracentrifuge using a TLS-55 rotor (Beckman Instruments, Fullerton, Calif., USA). Gradients were fractionated by puncturing the tube bottom with a 20 ga. needle and collecting drops in a 96 well microtiter plate. 3 μl aliquots were analyzed on a 1% agarose mini-gel. Fractions containing the protein C DNA fragment were pooled and ethanol precipitated overnight at -20°C. in 0.3M sodium acetate. DNA pellets were resuspended in 50-100 μl UHP water and quantitated by fluorimetry. The protein C expression unit was diluted in Dulbecco's phosphate buffered saline without calcium and magnesium (containing, per liter, 0.2 g KCl, 0.2 g KH₂ PO₄, 8.0 g NaCl, 1.15 g Na₂ HPO₄) or in TE (10 mM Tris-HCl, 1 mM EDTA pH 7.5). DNA concentration is adjusted to about 6 μg/ml, prior to injection into the eggs (.sup.˜ 2 pl total DNA solution per egg).

Recipient females of 6-8 weeks of age are prepared by mating B6CBAF1 females in natural estrus with vasectomized males. Females possessing copulation plugs are then kept for transfer of microinjected eggs.

Following birth of potential transgenic animals, tail biopsies are taken, under anesthesia, at four weeks of age. Tissue samples are placed in 2 ml of tail buffer (0.3 M Na acetate, 50 mM NaCl, 1.5 mM MgCl2, 10 mM Tris-HCl, pH 8.5, 0.5% NP40, 0.5% Tween 20) containing 200 lg/ml proteinase K (Boehringer Mannheim, Mannheim, Germany) and vortexed. The samples are shaken (250 rpm) at 55°-60° C. for 3 hours to overnight. DNA prepared from biopsy samples is examined for the presence of the injected constructs by PCR and Southern blotting. The digested tissue is vigorously vortexed, and 5 ll aliquots are placed in 0.5 ml microcentrifuge tubes. Positive and negative tail samples are included as controls. Forty ll of silicone oil (BDH, Poole, UK) is added to each tube, and the tubes are briefly centrifuged. The tubes are incubated in the heating block of a thermal cycler (e.g. Omni-gene, Hybaid, Teddington, UK) to 95° C. for 10 minutes. Following this, each tube has a 45 μl aliquot of PCR mix added such that the final composition of each reaction mix is: 50 mM KCl; 2 mM MgCl₂ ; 10 mM Tris-HCl (pH 8.3); 0.01% gelatin; 0.1% NP40, 10% DMSO; 500 nM each primer, 200 μM dNTPs; 0.02 U/μl Taq polymerase (Boehringer Mannheim, Mannheim, Germany). The tubes are then cycled through 30 repeated temperature changes as required by the particular primers used. The primers may be varied but in all cases must target the BLG promoter region. This is specific for the injected DNA fragments because the mouse does not have a BLG gene. Twelve μl of 5× loading buffer containing Orange G marker dye (0.25% Orange G (Sigma) 15% Ficoll type 400 (Pharmacia Biosystems Ltd., Milton Keynes, UK)) is then added to each tube, and the reaction mixtures are electrophoresed on a 1.6% agarose gel containing ethidium bromide (Sigma) until the marker dye has migrated 2/3 of the length of the gel. The gel is visualized with a UV light source emitting a wavelength of 254 nm. Transgenic mice having one or more of the injected DNA fragments are identified by this approach.

Positive tail samples are processed to obtain pure DNA. The DNA samples are screened by Southern blotting using a BLG promoter probe (nucleotides 2523-4253 of SEQ ID NO: 7).

Southern blot analysis of transgenic mice prepared essentially as described above demonstrated that approximately 10% of progeny contained protein C sequences. Examination of milk from positive animals by reducing SDS polyacrylamide gel electrophoresis demonstrated the presence of protein C at concentrations up to 1 mg/ml.

Example 4

Donor ewes are treated with an intravaginal progesterone-impregnated sponge (CHRONOGEST Goat Sponge, Intervet, Cambridge, UK) on day 0. Sponges are left in situ for ten or twelve days.

Superovulation is induced by treatment of donor ewes with a total of one unit of ovine follicle stimulating hormone (OFSH) (OVAGEN, Horizon Animal Reproduction Technology Pty. Ltd., New Zealand) administered in eight intramuscular injections of 0.125 units per injection starting at 5:00 pm on day -4 and ending at 8:00 am on day 0. Donors are injected intramuscularly with 0.5 ml of a luteolytic agent (ESTRUMATE, Vet-Drug) on day -4 to cause regression of the corpus luteum, to allow return to estrus and ovulation. To synchronize ovulation, the donor animals are injected intramuscularly with 2 ml of a synthetic releasing hormone analog (RECEPTAL, Vet-Drug) at 5:00 pm on day 0.

Donors are starved of food and water for at least 12 hours before artificial insemination (A.I.). The animals are artificially inseminated by intrauterine laparoscopy under sedation and local anesthesia on day 1. Either xylazine (ROMPUN, Vet-Drug) at a dose rate of 0.05-0.1 ml per 10 kg bodyweight or ACP injection 10 mg/ml (Vet-Drug) at a dose rate of 0.1 ml per 10 kg bodyweight is injected intramuscularly approximately fifteen minutes before A.I. to provide sedation. A.I. is carried out using freshly collected semen from a Poll Dorset ram. Semen is diluted with equal parts of filtered phosphate buffered saline, and 0.2 ml of the diluted semen is injected per uterine horn. Immediately pre- or post-A.I., donors are given an intramuscular injection of AMOXYPEN (Vet-Drug).

Fertilized eggs are recovered on day 2 following starvation of donors of food and water from 5:00 pm on day 1. Recovery is carried out under general anesthesia induced by an intravenous injection of 5% thiopentone sodium (INTRAVAL SODIUM, Vet-Drug) at a dose rate of 3 ml per 10 kg bodyweight. Anesthesia is maintained by inhalation of 1-2% Halothane/O₂ /N₂ O. To recover the fertilized eggs, a laparotomy incision is made, and the uterus is exteriorized. The eggs are recovered by retrograde flushing of the oviducts with Ovum Culture Medium (Advanced Protein Products, Brierly Hill, West Midlands, UK) supplemented with bovine serum albumin of New Zealand origin. After flushing, the uterus is returned to the abdomen, and the incision is closed. Donors are allowed to recover post-operatively or are euthanized. Donors that are allowed to recover are given an intramuscular injection of Amoxypen L.A. at the manufacturer's recommended dose rate immediately pre- or post-operatively.

Plasmids containing the protein C DNA are digested with Mlu I, and the expression unit fragments are recovered and purified on sucrose density gradients. The fragment concentrations are determined by fluorimetry and diluted in Dulbecco's phosphate buffered saline without calcium and magnesium or TE as described above. The concentration is adjusted to 6 μg/ml, and approximately 2 pl of the mixture is microinjected into one pronucleus of each fertilized eggs with visible pronuclei.

All fertilized eggs surviving pronuclear microinjection are cultured in vitro at 38.5° C. in an atmosphere of 5% CO₂ :5% O2:90% N₂ and about .sub.˜ 100% humidity in a bicarbonate buffered synthetic oviduct medium (see Table) supplemented with 20% v/v vasectomized ram serum. The serum may be heat inactivated at 56° C. for 30 minutes and stored frozen at -20° C. prior to use. The fertilized eggs are cultured for a suitable period of time to allow early embryo mortality (caused by the manipulation techniques) to occur. These dead or arrested embryos are discarded. Embryos having developed to 5 or 6 cell divisions are transferred to synchronized recipient ewes.

                  TABLE     ______________________________________     Synthetic Oviduct Medium     ______________________________________     Stock A (Lasts 3 Months)     NaCl              6.29        g     KCl               0.534       g     KH.sub.2 PO.sub.4 0.162       g     MgSO.sub.4.7H.sub.2 O                       0.182       g     Penicillin        0.06        g     Sodium Lactate 60% syrup                       0.6         mls     Super H.sub.2 O   99.4        mls     Stock B (Lasts 2 weeks)     NaHCO.sub.3       0.21        g     Phenol red        0.001       g     Super H.sub.2 O   10          mls     Stock C (Lasts 2 weeks)     Sodium Pyruvate   0.051       g     Super H.sub.2 O   10          mls     Stock D (Lasts 3 months)     CaCl2.2H.sub.2 O  0.262       g     Super H.sub.2 O   10          mls     Stock E (Lasts 3 months)     Hepes             0.651       g     Phenol red        0.001       g     Super H.sub.2 O   10          mls     To make up 10 mls of Bicarbonate Buffered Medium     STOCK A           1           ml     STOCK B           1           ml     STOCK C           0.07        ml     STOCK D           0.1         ml     Super H.sub.2 O   7.83        ml     To make up 10 mls of HEPES Buffered Medium     STOCK A           1           ml     STOCK B           0.2         ml     STOCK C           0.07        ml     STOCK D           0.1         ml     STOCK E           0.8         ml     Super H2O         7.83        ml     ______________________________________      Osmolarity should be 265-285 mOsm.      Add 2.5 ml of heat inactivated sheep serum and filter sterilize.

Recipient ewes are treated with an intravaginal progesterone-impregnated sponge (Chronogest Ewe Sponge or Chronogest Ewe-Lamb Sponge, Intervet) left in situ for 10 or 12 days. The ewes are injected intramuscularly with 1.5 ml (300 iu) of a follicle stimulating hormone substitute (P.M.S.G., Intervet) and with 0.5 ml of a luteolytic agent (Estrumate, Coopers Pitman-Moore) at sponge removal on day -1. The ewes are tested for estrus with a vasectomized ram between 8:00 am and 5:00 pm on days 0 and 1.

Embryos surviving in vitro culture are returned to recipients (starved from 5:00 pm on day 5 or 6) on day 6 or 7. Embryo transfer is carried out under general anesthesia as described above. The uterus is exteriorized via a laparotomy incision with or without laparoscopy. Embryos are returned to one or both uterine horns only in ewes with at least one suitable corpora lutea. After replacement of the uterus, the abdomen is closed, and the recipients are allowed to recover. The animals are given an intramuscular injection of Amoxypen L.A. at the manufacturer's recommended dose rate immediately pre- or post-operatively.

Lambs are identified by ear tags and left with their dams for rearing. Ewes and lambs are either housed and fed complete diet concentrates and other supplements and or ad lib. hay, or are let out to grass.

Within the first week of life (or as soon thereafter as possible without prejudicing health), each lamb is tested for the presence of the heterologous DNA by two sampling procedures. Following tail biopsy, within a week, a 10 ml blood sample is taken from the jugular vein into an EDTA vacutainer. Tissue samples are taken by tail biopsy as soon as possible after the tail has become desensitized after the application of a rubber elastrator ring to its proximal third (usually within 200 minutes after "tailing") The tissue is placed immediately in a solution of tail buffer. Tail samples are kept at room temperature and analyzed on the day of collection. All lambs are given an intramuscular injection of Amoxypen L.A at the manufacturer's recommended dose rate immediately post-biopsy, and the cut end of the tail is sprayed with an antibiotic spray.

DNA is extracted from sheep blood by first separating white blood cells. A 10 ml sample of blood is diluted in 20 ml of Hank's buffered saline (HBS; obtained from Sigma Chemical Co.). Ten ml of the diluted blood is layered over 5 ml of Histopaque (Sigma) in each of two 15 ml screw-capped tubes. The tubes are centrifuged at 3000 rpm (2000×g max.), low brake for 15 minutes at room temperature. White cell interfaces are removed to a clean 15 ml tube and diluted to 15 ml in HBS. The diluted cells are spun at 3000 rpm for 10 minutes at room temperature, and the cell pellet is recovered and resuspended in 2-5 ml of tail buffer.

To extract DNA from the white cells, 10% SDS is added to the resuspended cells to a final concentration of 1%, and the tube is inverted to mix the solution. One mg of fresh proteinase K solution is added, and the mixture is incubated overnight at 45° C. DNA is extracted using an equal volume of phenol/chloroform (×3) and chloroform/isoamyl alcohol (×1). The DNA is then precipitated by adding 0.1 volume of 3 M NaOAc and 2 volumes of ethanol, and the tube is inverted to mix. The precipitated DNA is spooled out using a clean glass rod with a sealed end. The spool is washed in 70% ethanol, and the DNA is allowed to partially dry, then is redissolved in TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.5).

DNA samples from blood and tail are analyzed by Southern blotting using probes for the BLG promoter region and the protein C coding regions.

Example 5

A founder female animal, designated 30851, which is transgenic for both BLG and pCORP9 was generated. She has given rise to two sons and a transgenic daughter, designated 40387. Recombinant transgenic protein C was purified from milk (from 30851) by a single chromatography step using a calcium-dependent monoclonal antibody affinity column. Briefly, the milk samples were pooled up to a volume of 40 ml. Two volumes of ice-cold 1×TBS (50 mM Tris-HCl, 150 mM NaCl pH 6.5) and 200 mM EDTA, pH 6.5 were added to solubilise the caseins. The EDTA-treated milk solution was centrifuged at 15,000 rpm for 30 minutes at 4° C. in a JA20 rotor (Beckman Instruments, Irvine, Calif.). After centrifugation, the upper lipid phase and the small pellet were discarded.

The EDTA-treated milk was diluted with an equal volume of ice-cold 1×TBS and 133 mM CaCl₂ while stirring. A cloudy precipitate formed upon addition of the CaCl₂. The pH was quickly adjusted by addition of a few drops of 4 M NaOH, and the precipitate was redissolved. Any remaining insoluble material was removed by filtration through a 0.45 μm filter.

The optical density of the solubilised milk was measured at 280 nm, and the protein concentration was calculated. The milk was diluted to a protein concentration of 10 mg/ml using 1×TBS containing CaCl₂ to give a final Ca⁺⁺ concentration of 25 mM. The milk was used to resuspend antibody-Sepharose that carried the immobilized Ca⁺⁺ -dependent monoclonal antibody PCL-2, and had been washed in 1×TBS and 25 mM CaCl₂. PCL-2 is a monoclonal antibody that binds single chain and two chain protein C, whether or not they are gamma-carboxylated. The milk-Sepharose mixture was incubated overnight at 4° C.

The matrix was washed twice in batch with 1×TBS and 25 mM CaCl₂ and packed into a glass column. The resin was washed at a flow rate of 1 ml/min with a calcium containing buffer and a stable baseline was achieved before the bound protein was eluted with an isocratic elution using 1×TBS and 25 mM EDTA, pH 6.5. Fractions containing protein C were pooled and concentrated to approximately 1 ml using an Amicon ultrafiltration unit with a 10 kDa cut-off membrane (Amicon, Danvers, Mass.).

The monoclonal antibody, PCL-2, was coupled to the activated Sepharose 4B as follows: 1 g (3.5 ml of gel) of cyanogen bromide activated Sepharose 4B (Pharmacia LKB Biotechnology, Piscataway, N.J.) was swollen for 15 minutes in 1 mM HCl. The swollen gel was resuspended in 0.1 M NaHCO₃, 0.5 M NaCl pH 8.3 and washed several times. The washed gel was resuspended in 11 ml of monoclonal antibody solution (PCL-2, 3.5 mg/ml in bicarbonate buffer pH 8.3) with a coupling ratio of approximately 10 mg/ml gel. Coupling was allowed to proceed for 2 h at room temperature on a rotary mixer, and the gel was recovered by gentle centrifugation. The monoclonal supernatant was removed and replaced by 1 M ethanolamine in order to block any remaining sites on the Sepharose. Blocking was performed overnight at 4° C. Excess adsorbed protein was removed by sequential acid and alkali washes (0.1 M acetate, 0.5 M NaCl pH 4.0; 0.1 M NaHCO₃, 0.5 M NaCl pH 8.3), and the coupled gel was stored in 50 mM Tris-HCl, 150 mM NaCl pH 6.5, 0.02% azide.

Example 6

Samples of purified recombinant transgenic protein C were compared with plasma-derived protein C and a plasma-derived activated protein C (APC) preparations. Samples were run on SDS PAGE 4-20% acrylamide gradient gels under reducing conditions and silver stained for protein.

The plasma-derived material shows the presence of a heavy-chain doublet around 44 kDa (FIG. 1, Lane 1). This has been reported to be due to partial occupancy of the three possible N-linked glycosylation sites on the molecule. A similar doublet, although of a slightly lower mass presumably due to some subtle change in glycosylation profile, has also been seen with the transgenic protein C. The light chain was visible around 22 kDa for both preparations. Significantly, in the case of the plasma-derived material uncleaved single-chain was clearly visible above the heavy chain doublet. Plasma-derived protein normally contained 5-10 percent of this inactive material. In contrast, the transgenic protein C contains no obvious single chain by this gel analysis. Therefore, it contains less than a few percent at most of inactive material. This most likely reflects the increased efficiency of cleavage of the modified inter-chain site. In further support of this observation no single chain was visible by direct western blot analysis of transgenic sheep milk (40387, expression level 300 μg/ml).

The purified transgenic protein C was further characterized as follows:

A. ELISA

An enzyme-linked immunosorbent assay (ELISA) for protein C was done as follows: Affinity-purified polyclonal antibody to human protein C (100 μl of 1 μg/ml in 0.1 M Na₂ CO₃, pH 9.6) was added to each well of a 96-well microtiter plate, and the plates were incubated overnight at 4° C. The wells were then washed three times with phosphate buffered saline (PBS) containing 0.05% Tween-20 and incubated with 100 μl of 1% bovine serum albumin (BSA), 0.05% Tween-20 in PBS at 4° C. overnight. The plates were then rinsed several times with PBS, air dried and stored at 4° C. To assay samples, 100 μl of each sample was incubated for 1 h at 37° C. with a biotin-conjugated sheep polyclonal antibody to protein C (30 ng/ml) in PBS containing 1% BSA and 0.05% Tween-20. After incubation, the wells were rinsed with PBS, and alkaline phosphatase activity was measured by the addition of 100 μl of phosphatase substrate (Sigma, St. Louis, Mo.) in 10% diethanolamine, pH 9.8, containing 0.3 mM MgCl₂. The absorbance at 405 nm was read on a microtiter plate reader. Quantitation was by comparison with a standard curve using plasma-derived protein C quantitated by amino acid analysis.

B. Amino-Terminal Sequencing

Amino-terminal sequencing of the transgenic material was performed to ascertain the extent of prosequence removal and to evaluate the presence of gamma-carboxylation. There were three possible N-terminal sequences of protein C. These were: 1) Prosequence which directs gamma-carboxylation and could have remained on the light chain if the first cleavage site was incompletely processed, 2) the light chain and 3) the heavy chain. N-terminal sequencing of protein C obtained from transgenic milk should have contained only the latter two sequences if correct processing had occurred at both of the cleavage sites. Amino-terminal sequencing would have also been expected to reveal the presence of gamma-carboxylation in the light chain. There are nine sites of carboxylation in the first twenty-nine amino acids of the light chain. On an analysis of released amino acids, the PTH-gamma carboxylic acid derivatives eluted from the HPLC column in the break-through and could therefore be analyzed. Thus, a gamma carboxylic acid showed up on the amino-terminal sequence as a space rather than a glutamic acid.

The yields of amino acids in pmol released from the sequencing of approximately 27 pmol (1.4 μl) of purified transgenic protein C corresponded well to those expected for an equimolar mixture of light and heavy chains, and no obvious sequence was discernible for the prosequence. Moreover, no other aberrant sequences were detected, thus indicating a lack of inappropriate proteolytic cleavages.

As stated previously, gamma-carboxylated glutamate residues were expected to sequence as blanks using standard instrument conditions. However, sequencing protein C gives a double sequence which must be deconvoluted using knowledge of the expected light and heavy chain sequences. Normally, if the light chain alone were sequenced the gla residues at positions six and seven would appear as blanks. However when sequenced as intact protein C, the heavy chain sequence contains a glutamate residue at position six. Therefore, the only indirect confirmation of the presence of a gla residue in the light chain was the absence of glutamate at position seven which was not `over written` by a glutamate in the heavy chain (FIG. 2). Two other indirect confirmations of the presence of gamma carboxylation of the transgenic product are described below.

C. Mass Analysis of the Purified Light Chain

The protein sequence of the transgenic-derived protein C precursor had been modified with an Arg-Arg-Lys-Arg (SEQ ID NO: 20) cleavage site between the light and heavy chains to promote more efficient cleavage of the single chain to 2-chain form. Western blot analysis of the transgenic protein C milk and examination of the purified protein C on reducing gels had already confirmed that efficient cleavage had occurred. Normally during secretion, but after cleavage of the plasma-derived material, the two basic amino acids at the carboxy-terminus of the light chain are trimmed back by a basic carboxypeptide. Establishing whether the carboxy-terminus of the transgenic protein C light chain had been processed to remove the two extra basic amino acids introduced by this modification, as well as the two natural ones, was achieved by measuring the mass of the purified light chain in a quadropole instrument using on-line liquid chromatography and electro-spray ionization. In order to achieve this, all of the cysteine residues of protein C were reduced and alkylated, and then the two chains were separated by reversed-phase chromatography.

C1. Reductive Alkylation

Because protein C is heavily cross-linked for a molecule of approximately 52 kDa, with twelve disulfide bridges (17 of the 24 cysteines involved are in the light chain), it was necessary to reductively alkylate the entire protein before attempting to separate the chains by reversed-phase chromatography. In view of the large number of cysteines in the light chain, alkylatation was done with iodoacetamide, in place of the more commonly used vinyl pyridine, to prevent the molecule from becoming excessively hydrophobic.

The transgenic protein C material (6 nmol of protein or 144 pmol of thiol) was reductively alkylated as follows: 0.5 mg of protein C (by ELISA) in 0.5 ml of TBS was added to 50 μl of 1 M Tris pH 8.0, 450 μl water, 570 mg guanidinium chloride, and 10 μl at 50 mg/ml DTT (0.3 μmol representing a 20 fold excess of added thiol over cysteine thiol. The mixture was incubated for 2 hours at 37° C. After incubation, 20 μl at 120 mg/ml iodoacetamide (0.6 M representing a 2 fold excess over DTT on a molar basis) was added, and the mixture was incubated in the dark for one hour at 4° C. The reaction was quenched by adding 50 μl at 50 mg/ml DTT representing a 2.5 fold excess over iodoacetamide. The sample (final volume 1.5 ml) was stored at -20° C. until analysis.

D. Purification of the Light Chain

Purification of protein C light chain was achieved using a large pore polystyrene column with divinyl benzene interactive groups (PLRP-S, 4000Å, 8 μm, 2.1 mm ID: Polymer Laboratories, Shropshire, UK). The optimum conditions for separation of the heavy and light chains were determined to be: solvent A (0.1% TFA) and solvent B (100% acetonitrile) at a flow of 0.5 ml/min with a detector wavelength of 215 nm and a gradient of 30 to 60% solvent B over 60 min.

Fractions were collected across the eluted peaks, and samples (10 μl ) were analyzed by SDS PAGE on 4-20% gradient acrylamide gels under non-reducing conditions. The light chain (fractions 3 to 5) was completely resolved from both the heavy chain (fractions 7 to 9) and a single fraction (6) which contained a mixture of heavy chain and what appeared to be unglycosylated light chain.

A sample containing fully resolved light chain was prepared for deglycosylation by centrifugal evaporation under reduced pressure at room temperature. Deglycosylation was carried out using peptide N-glycanase (PNGase; Oxford Glycosystems, Oxford, UK). The protein sample was redissolved in 50 μl of buffer and incubated overnight with 1 unit (5 μl) PNGase, according to manufacturer's specifications.

The light chain was purified from reduced and alkylated plasma-derived protein C by the same method and deglycosylated for further analysis.

E. Analysis by Mass Spectroscopy

Samples of purified light chain were subjected to mass analysis using a liquid chromatography--electrospray interface to a Sciex Quadropole Mass Analyser (Sciex/Perkin Elmer, Toronto, Calif.). The LC system used a 0.5 mm ID column packed with PLRP-S 4000Å, 8 μm resin (Polymer Laboratories). The solvent system contained buffer A (0.1% formic acid), buffer B (0.1% formic acid and a 5:2 (v/v) mixture of ethanol to propan-1-ol). The gradient used was from 5-60% buffer B over 35 minutes at a flow rate of 25 μl per minute. The outflow of the column was linked via a UV detector to the mass spectrometer which was run in positive-ion mode.

The purified and deglycosylated transgenic light chain was analyzed and gave a relatively weak spectrum which was reconstructed to give two components with masses of 18,911.0 and 18,971.0. The plasma light chain was also analyzed and gave a stronger signal with a single major component. The spectrum of the plasma light chain was reconstructed to give a single mass of 18,970.0.

The predicted mass for the light chain carrying nine gamma-carboxy glutamic acids, one β-hydroxy aspartic acid and seventeen carbamidomethyl cysteine residues and ending with Leu₁₅₅ was 18966.9723, which is very close to the masses detected for the transgenic (18,971.0) and plasma-derived (18,970.0) light chains. The small differences in mass were well within the accuracy limitations for this instrument, particularly with the LC delivery. This shows that the mass of the redirectively-alkylated and deglycosylated transgenic light chain is essentially identical to that for the plasma-derived protein C. This implies that both molecules have undergone the same post-translational modifications and that the transgenic material is fully gamma carboxylated, has had all four basic amino acids trimmed back from the carboxy-terminus of the light chain and has single β hydroxy-alanine.

F. Activity Measurements

The activity of the transgenic protein C was compared with that of the plasma-derived material in a coagulation assay. First each sample of protein C, quantitated by amino acid composition analysis, was activated by incubation with Protac, a snake venom (American Diagnostica Inc, Greenwich, Conn.) at a venom to protein ratio of 1 Unit Protac: 10 μg protein C for 60 minutes at 37° C. Aliquots of the activated material were then compared for their ability to prolong the clotting time of protein C depleted human plasma (Diagnostic Reagents Ltd) in the presence of activated partial thromboplastin time reagent--cephalin from rabbit brain (Sigma) and calcium using a mechanical coagulometer (Diagnostica Stago, Asmieres, FR) A comparison of clotting times with various additions of transgenic and plasma-derived protein C (FIG. 3) shows that the two preparations had the same anti-coagulant activity per mg of protein.

In summary, results show that the sheep-derived transgenic protein C is correctly post-translationally processed, with respect to gamma-carboxylation and probably beta-hydroxylation, and has anticoagulant activity fully equivalent to a high quality purified plasma standard. The results demonstrate that the C-terminal processing of the light chain, with the modified RRKR cleavage site rather than the naturally occurring KR site, has the two extra basic amino acids removed along with the natural ones.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

    __________________________________________________________________________     #             SEQUENCE LISTING     - (1) GENERAL INFORMATION:     -    (iii) NUMBER OF SEQUENCES: 25     - (2) INFORMATION FOR SEQ ID NO:1:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 11725 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: double               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA (genomic)     -     (ix) FEATURE:               (A) NAME/KEY: CDS               (B) LOCATION: join(3520..3 - #630, 5093..5117, 5210..5347,     5450                    ..5584, 8 - #253..8395, 9269..9386, 10516..11102)     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:     - AGTGAATCTG GGCGAGTAAC ACAAAACTTG AGTGTCCTTA CCTGAAAAAT AG - #AGGTTAGA       60     - GGGATGCTAT GTGCCATTGT GTGTGTGTGT TGGGGGTGGG GATTGGGGGT GA - #TTTGTGAG      120     - CAATTGGAGG TGAGGGTGGA GCCCAGTGCC CAGCACCTAT GCACTGGGGA CC - #CAAAAAGG      180     - AGCATCTTCT CATGATTTTA TGTATCAGAA ATTGGGATGG CATGTCATTG GG - #ACAGCGTC      240     - TTTTTTCTTG TATGGTGGCA CATAAATACA TGTGTCTTAT AATTAATGGT AT - #TTTAGATT      300     - TGACGAAATA TGGAATATTA CCTGTTGTGC TGATCTTGGG CAAACTATAA TA - #TCTCTGGG      360     - CAAAAATGTC CCCATCTGAA AAACAGGGAC AACGTTCCTC CCTCAGCCAG CC - #ACTATGGG      420     - GCTAAAATGA GACCACATCT GTCAAGGGTT TTGCCCTCAC CTCCCTCCCT GC - #TGGATGGC      480     - ATCCTTGGTA GGCAGAGGTG GGCTTCGGGC AGAACAAGCC GTGCTGAGCT AG - #GACCAGGA      540     - GTGCTAGTGC CACTGTTTGT CTATGGAGAG GGAGGCCTCA GTGCTGAGGG CC - #AAGCAAAT      600     - ATTTGTGGTT ATGGATTAAC TCGAACTCCA GGCTGTCATG GCGGCAGGAC GG - #CGAACTTG      660     - CAGTATCTCC ACGACCCGCC CCTGTGAGTC CCCCTCCAGG CAGGTCTATG AG - #GGGTGTGG      720     - AGGGAGGGCT GCCCCCGGGA GAAGAGAGCT AGGTGGTGAT GAGGGCTGAA TC - #CTCCAGCC      780     - AGGGTGCTCA ACAAGCCTGA GCTTGGGGTA AAAGGACACA AGGCCCTCCA CA - #GGCCAGGC      840     - CTGGCAGCCA CAGTCTCAGG TCCCTTTGCC ATGCGCCTCC CTCTTTCCAG GC - #CAAGGGTC      900     - CCCAGGCCCA GGGCCATTCC AACAGACAGT TTGGAGCCCA GGACCCTCCA TT - #CTCCCCAC      960     - CCCACTTCCA CCTTTGGGGG TGTCGGATTT GAACAAATCT CAGAAGCGGC CT - #CAGAGGGA     1020     - GTCGGCAAGA ATGGAGAGCA GGGTCCGGTA GGGTGTGCAG AGGCCACGTG GC - #CTATCCAC     1080     - TGGGGAGGGT TCCTTGATCT CTGGCCACCA GGGCTATCTC TGTGGCCTTT TG - #GAGCAACC     1140     - TGGTGGTTTG GGGCAGGGGT TGAATTTCCA GGCCTAAAAC CACACAGGCC TG - #GCCTTGAG     1200     - TCCTGGCTCT GCGAGTAATG CATGGATGTA AACATGGAGA CCCAGGACCT TG - #CCTCAGTC     1260     - TTCCGAGTCT GGTGCCTGCA GTGTACTGAT GGTGTGAGAC CCTACTCCTG GA - #GGATGGGG     1320     - GACAGAATCT GATCGATCCC CTGGGTTGGT GACTTCCCTG TGCAATCAAC GG - #AGACCAGC     1380     - AAGGGTTGGA TTTTTAATAA ACCACTTAAC TCCTCCGAGT CTCAGTTTCC CC - #CTCTATGA     1440     - AATGGGGTTG ACAGCATTAA TAACTACCTC TTGGGTGGTT GTGAGCCTTA AC - #TGAAGTCA     1500     - TAATATCTCA TGTTTACTGA GCATGAGCTA TGTGCAAAGC CTGTTTTGAG AG - #CTTTATGT     1560     - GGACTAACTC CTTTAATTCT CACAACACCC TTTAAGGCAC AGATACACCA CG - #TTATTCCA     1620     - TCCATTTTAC AAATGAGGAA ACTGAGGCAT GGAGCAGTTA AGCATCTTGC CC - #AACATTGC     1680     - CCTCCAGTAA GTGCTGGAGC TGGAATTTGC ACCGTGCAGT CTGGCTTCAT GG - #CCTGCCCT     1740     - GTGAATCCTG TAAAAATTGT TTGAAAGACA CCATGAGTGT CCAATCAACG TT - #AGCTAATA     1800     - TTCTCAGCCC AGTCATCAGA CCGGCAGAGG CAGCCACCCC ACTGTCCCCA GG - #GAGGACAC     1860     - AAACATCCTG GCACCCTCTC CACTGCATTC TGGAGCTGCT TTCTAGGCAG GC - #AGTGTGAG     1920     - CTCAGCCCCA CGTAGAGCGG GCAGCCGAGG CCTTCTGAGG CTATGTCTCT AG - #CGAACAAG     1980     - GACCCTCAAT TCCAGCTTCC GCCTGACGGC CAGCACACAG GGACAGCCCT TT - #CATTCCGC     2040     - TTCCACCTGG GGGTGCAGGC AGAGCAGCAG CGGGGGTAGC ACTGCCCGGA GC - #TCAGAAGT     2100     - CCTCCTCAGA CAGGTGCCAG TGCCTCCAGA ATGTGGCAGC TCACAAGCCT CC - #TGCTGTTC     2160     - GTGGCCACCT GGGGAATTTC CGGCACACCA GCTCCTCTTG GTAAGGCCAC CC - #CACCCCTA     2220     - CCCCGGGACC CTTGTGGCCT CTACAAGGCC CTGGTGGCAT CTGCCCAGGC CT - #TCACAGCT     2280     - TCCACCATCT CTCTGAGCCC TGGGTGAGGT GAGGGGCAGA TGGGAATGGC AG - #GAATCAAC     2340     - TGACAAGTCC CAGGTAGGCC AGCTGCCAGA GTGCCACACA GGGGCTGCCA GG - #GCAGGCAT     2400     - GCGTGATGGC AGGGAGCCCC GCGATGACCT CCTAAAGCTC CCTCCTCCAC AC - #GGGGATGG     2460     - TCACAGAGTC CCCTGGGCCT TCCCTCTCCA CCCACTCACT CCCTCAACTG TG - #AAGACCCC     2520     - AGGCCCAGGC TACCGTCCAC ACTATCCAGC ACAGCCTCCC CTACTCAAAT GC - #ACACTGGC     2580     - CTCATGGCTG CCCTGCCCCA ACCCCTTTCC TGGTCTCCAC AGCCAACGGG AG - #GAGGCCAT     2640     - GATTCTTGGG GAGGTCCGCA GGCACATGGG CCCCTAAAGC CACACCAGGC TG - #TTGGTTTC     2700     - ATTTGTGCCT TTATAGAGCT GTTTATCTGC TTGGGACCTG CACCTCCACC CT - #TTCCCAAG     2760     - GTGCCCTCAG CTCAGGCATA CCCTCCTCTA GGATGCCTTT TCCCCCATCC CT - #TCTTGCTC     2820     - ACACCCCCAA CTTGATCTCT CCCTCCTAAC TGTGCCCTGC ACCAAGACAG AC - #ACTTCACA     2880     - GAGCCCAGGA CACACCTGGG GACCCTTCCT GGGTGATAGG TCTGTCTATC CT - #CCAGGTGT     2940     - CCCTGCCCAA GGGGAGAAGC ATGGGGAATA CTTGGTTGGG GGAGGAAAGG AA - #GACTGGGG     3000     - GGATGTGTCA AGATGGGGCT GCATGTGGTG TACTGGCAGA AGAGTGAGAG GA - #TTTAACTT     3060     - GGCAGCCTTT ACAGCAGCAG CCAGGGCTTG AGTACTTATC TCTGGGCCAG GC - #TGTATTGG     3120     - ATGTTTTACA TGACGGTCTC ATCCCCATGT TTTTGGATGA GTAAATTGAA CC - #TTAGAAAG     3180     - GTAAAGACAC TGGCTCAAGG TCACACAGAG ATCGGGGTGG GGTTCACAGG GA - #GGCCTGTC     3240     - CATCTCAGAG CAAGGCTTCG TCCTCCAACT GCCATCTGCT TCCTGGGGAG GA - #AAAGAGCA     3300     - GAGGACCCCT GCGCCAAGCC ATGACCTAGA ATTAGAATGA GTCTTGAGGG GG - #CGGAGACA     3360     - AGACCTTCCC AGGCTCTCCC AGCTCTGCTT CCTCAGACCC CCTCATGGCC CC - #AGCCCCTC     3420     - TTAGGCCCCT CACCAAGGTG AGCTCCCCTC CCTCCAAAAC CAGACTCAGT GT - #TCTCCAGC     3480     - AGCGAGCGTG CCCACCAGGT GCTGCGGATC CGCAAACGT GCC AAC TCC - # TTC CTG     3534     - GAG GAG CTC CGT CAC AGC AGC CTG GAG CGG GA - #G TGC ATA GAG GAG ATC     3582     - TGT GAC TTC GAG GAG GCC AAG GAA ATT TTC CA - #A AAT GTG GAT GAC ACA     3630     - GTAAGGCCAC CATGGGTCCA GAGGATGAGG CTCAGGGGCG AGCTGGTAAC CA - #GCAGGGGC     3690     - CTCGAGGAGC AGGTGGGGAC TCAATGCTGA GGCCCTCTTA GGAGTTGTGG GG - #GTGGCTGA     3750     - GTGGAGCGAT TAGGATGCTG GCCCTATGAT GTCGGCCAGG CACATGTGAC TG - #CAAGAAAC     3810     - AGAATTCAGG AAGAAGCTCC AGGAAAGAGT GTGGGGTGAC CCTAGGTGGG GA - #CTCCCACA     3870     - GCCACAGTGT AGGTGGTTCA GTCCACCCTC CAGCCACTGC TGAGCACCAC TG - #CCTCCCCG     3930     - TCCCACCTCA CAAAGAGGGG ACCTAAAGAC CACCCTGCTT CCACCCATGC CT - #CTGCTGAT     3990     - CAGGGTGTGT GTGTGACCGA AACTCACTTC TGTCCACATA AAATCGCTCA CT - #CTGTGCCT     4050     - CACATCAAAG GGAGAAAATC TGATTGTTCA GGGGGTCGGA AGACAGGGTC TG - #TGTCCTAT     4110     - TTGTCTAAGG GTCAGAGTCC TTTGGAGCCC CCAGAGTCCT GTGGACGTGG CC - #CTAGGTAG     4170     - TAGGGTGAGC TTGGTAACGG GGCTGGCTTC CTGAGACAAG GCTCAGACCC GC - #TCTGTCCC     4230     - TGGGGATCGC TTCAGCCACC AGGACCTGAA AATTGTGCAC GCCTGGGCCC CC - #TTCCAAGG     4290     - CATCCAGGGA TGCTTTCCAG TGGAGGCTTT CAGGGCAGGA GACCCTCTGG CC - #TGCACCCT     4350     - CTCTTGCCCT CAGCCTCCAC CTCCTTGACT GGACCCCCAT CTGGACCTCC AT - #CCCCACCA     4410     - CCTCTTTCCC CAGTGGCCTC CCTGGCAGAC ACCACAGTGA CTTTCTGCAG GC - #ACATATCT     4470     - GATCACATCA AGTCCCCACC GTGCTCCCAC CTCACCCATG GTCTCTCAGC CC - #CAGCAGCC     4530     - TTGGCTGGCC TCTCTGATGG AGCAGGCATC AGGCACAGGC CGTGGGTCTC AA - #CGTGGGCT     4590     - GGGTGGTCCT GGACCAGCAG CAGCCGCCGC AGCAGCAACC CTGGTACCTG GT - #TAGGAACG     4650     - CAGACCCTCT GCCCCCATCC TCCCAACTCT GAAAAACACT GGCTTAGGGA AA - #GGCGCGAT     4710     - GCTCAGGGGT CCCCCAAAGC CCGCAGGCAG AGGGAGTGAT GGGACTGGAA GG - #AGGCCGAG     4770     - TGACTTGGTG AGGGATTCGG GTCCCTTGCA TGCAGAGGCT GCTGTGGGAG CG - #GACAGTCG     4830     - CGAGAGCAGC ACTGCAGCTG CATGGGGAGA GGGTGTTGCT CCAGGGACGT GG - #GATGGAGG     4890     - CTGGGCGCGG GCGGGTGGCG CTGGAGGGCG GGGGAGGGGC AGGGAGCACC AG - #CTCCTAGC     4950     - AGCCAACGAC CATCGGGCGT CGATCCCTGT TTGTCTGGAA GCCCTCCCCT CC - #CCTGCCCG     5010     - CTCACCCGCT GCCCTGCCCC ACCCGGGCGC GCCCCTCCGC ACACCGGCTG CA - #GGAGCCTG     5070     - ACGCTGCCCG CTCTCTCCGC AG CTG GCC TTC TGG TCC AAG - # CAC GTC  G     5117     - GTGAGTGCGT TCTAGATCCC CGGCTGGACT ACCGGCGCCC GCGCCCCTCG GG - #ATCTCTGG     5177     #CAG TGC TTG GTC     5229TTGTGTCGC AG  AC GGT GAC     - TTG CCC TTG GAG CAC CCG TGC GCC AGC CTG TG - #C TGC GGG CAC GGC ACG     5277     TGC ATC GAC GGC ATC GGC AGC TTC AGC TGC GA - #C TGC CGC AGC GGC TGG     5325     #GAGGTGGATG CTGGCGGGCG     5377GTGAGGGGGA     GCGGGGCGGG GCTGGGGCCG GGTTGGGGGC GCGGCACCAG CACCAGCTGC CC - #GCGCCCTC     5437     #TCT CTG GAC AAC GGC       5484 CTC AAT TGC     - GGC TGC ACG CAT TAC TGC CTA GAG GAG GTG GG - #C TGG CGG CGC TGT AGC     5532     - TGT GCG CCT GGC TAC AAG CTG GGG GAC GAC CT - #C CTG CAG TGT CAC CCC     5580     #TGCGGGGTGG       5634CAATACAT CGCCCAGGAA TCACGCTGGG     - GCAGGCCCCT GACGGGCGCG GCGCGGGGGG CTCAGGAGGG TTTCTAGGGA GG - #GAGCGAGG     5694     - AACAGAGTTG AGCCTTGGGG CAGCGGCAGA CGCGCCCAAC ACCGGGGCCA CT - #GTTAGCGC     5754     - AATCAGCCCG GGAGCTGGGC GCGCCCTCCG CTTTCCCTGC TTCCTTTCTT CC - #TGGCGTCC     5814     - CCGCTTCCTC CGGGCGCCCC TGCGACCTGG GGCCACCTCC TGGAGCGCAA GC - #CCAGTGGT     5874     - GGCTCCGCTC CCCAGTCTGA GCGTATCTGG GGCGAGGCGT GCAGCGTCCT CC - #TCCATGTA     5934     - GCCTGGCTGC GTTTTTCTCT GACGTTGTCC GGCGTGCATC GCATTTCCCT CT - #TTACCCCC     5994     - TTGCTTCCTT GAGGAGAGAA CAGAATCCCG ATTCTGCCTT CTTCTATATT TT - #CCTTTTTA     6054     - TGCATTTTAA TCAAATTTAT ATATGTATGA AACTTTAAAA ATCAGAGTTT TA - #CAACTCTT     6114     - ACACTTTCAG CATGCTGTTC CTTGGCATGG GTCCTTTTTT CATTCATTTT CA - #TAAAAGGT     6174     - GGACCCTTTT AATGTGGAAA TTCCTATCTT CTGCCTCTAG GGCATTTATC AC - #TTATTTCT     6234     - TCTACAATCT CCCCTTTACT TCCTCTATTT TCTCTTTCTG GACCTCCCAT TA - #TTCAGACC     6294     - TCTTTCCTCT AGTTTTATTG TCTCTTCTAT TTCCCATCTC TTTGACTTTG TG - #TTTTCTTT     6354     - CAGGGAACTT TCTTTTTTTT CTTTTTTTTT GAGATGGAGT TTCACTCTTG TT - #GTCCCAGG     6414     - CTGGAGTGCA ATGACGTGAT CTCAGCTCAC CACAACCTCC GCCTCCTGGA TT - #CAAGCGAT     6474     - TCTCCTGCCG CAGCCTCCCG AGTAGCTGGG ATTACAGGCA TGCGCCACCA CG - #CCCAGCTA     6534     - ATTTTGTGTT TTTAGTAGAG AAGGGGTTTC TCCGTGTTGG TCAAGCTGGT CT - #TGAACTCC     6594     - TGACCTCAGG TGATCCACCT GCCTTGGCCT CCTAAAGTGC TGGGATTACA GG - #CGTGAGCC     6654     - ACCGCGCCCA GCCTCTTTCA GGGAACTTTC TACAACTTTA TAATTCAATT CT - #TCTGCAGA     6714     - AAAAAATTTT TGGCCAGGCT CAGTAGCTCA GACCAATAAT TCCAGCACTT TG - #AGAGGCTG     6774     - AGGTGGGAGG ATTGCTTGAG CTTGGGAGTT TGAGACTAGC CTGGGCAACA CA - #GTGAGACC     6834     - CTGTCTCTAT TTTTAAAAAA AGTAAAAAAA GATCTAAAAA TTTAACTTTT TA - #TTTTGAAA     6894     - TAATTAGATA TTTCCAGGAA GCTGCAAAGA AATGCCTGGT GGGCCTGTTG GC - #TGTGGGTT     6954     - TCCTGCAAGG CCGTGGGAAG GCCCTGTCAT TGGCAGAACC CCAGATCGTG AG - #GGCTTTCC     7014     - TTTTAGGCTG CTTTCTAAGA GGACTCCTCC AAGCTCTTGG AGGATGGAAG AC - #GCTCACCC     7074     - ATGGTGTTCG GCCCCTCAGA GCAGGGTGGG GCAGGGGAGC TGGTGCCTGT GC - #AGGCTGTG     7134     - GACATTTGCA TGACTCCCTG TGGTCAGCTA AGAGCACCAC TCCTTCCTGA AG - #CGGGGCCT     7194     - GAAGTCCCTA GTCAGAGCCT CTGGTTCACC TTCTGCAGGC AGGGAGAGGG GA - #GTCAAGTC     7254     - AGTGAGGAGG GCTTTCGCAG TTTCTCTTAC AAACTCTCAA CATGCCCTCC CA - #CCTGCACT     7314     - GCCTTCCTGG AAGCCCCACA GCCTCCTATG GTTCCGTGGT CCAGTCCTTC AG - #CTTCTGGG     7374     - CGCCCCCATC ACGGGCTGAG ATTTTTGCTT TCCAGTCTGC CAAGTCAGTT AC - #TGTGTCCA     7434     - TCCATCTGCT GTCAGCTTCT GGAATTGTTG CTGTTGTGCC CTTTCCATTC TT - #TTGTTATG     7494     - ATGCAGCTCC CCTGCTGACG ACGTCCCATT GCTCTTTTAA GTCTAGATAT CT - #GGACTGGG     7554     - CATTCAAGGC CCATTTTGAG CAGAGTCGGG CTGACCTTTC AGCCCTCAGT TC - #TCCATGGA     7614     - GTATGCGCTC TCTTCTTGGC AGGGAGGCCT CACAAACATG CCATGCCTAT TG - #TAGCAGCT     7674     - CTCCAAGAAT GCTCACCTCC TTCTCCCTGT AATTCCTTTC CTCTGTGAGG AG - #CTCAGCAG     7734     - CATCCCATTA TGAGACCTTA CTAATCCCAG GGATCACCCC CAACAGCCCT GG - #GGTACAAT     7794     - GAGCTTTTAA GAAGTTTAAC CACCTATGTA AGGAGACACA GGCAGTGGGC GA - #TGCTGCCT     7854     - GGCCTGACTC TTGCCATTGG GTGGTACTGT TTGTTGACTG ACTGACTGAC TG - #ACTGGAGG     7914     - GGGTTTGTAA TTTGTATCTC AGGGATTACC CCCAACAGCC CTGGGGTACA AT - #GAGCCTTC     7974     - AAGAAGTTTA ACAACCTATG TAAGGACACA CAGCCAGTGG GTGATGCTGC CT - #GGTCTGAC     8034     - TCTTGCCATT CAGTGGCACT GTTTGTTGAC TGACTGACTG ACTGACTGGC TG - #ACTGGAGG     8094     - GGGTTCATAG CTAATATTAA TGGAGTGGTC TAAGTATCAT TGGTTCCTTG AA - #CCCTGCAC     8154     - TGTGGCAAAG TGGCCCACAG GCTGGAGGAG GACCAAGACA GGAGGGCAGT CT - #CGGGAGGA     8214     - GTGCCTGGCA GGCCCCTCAC CACCTCTGCC TACCTCAG  TG AAG T - #TC CCT TGT     8266     - GGG AGG CCC TGG AAG CGG ATG GAG AAG AAG CG - #C AGT CAC CTG AAA CGA     8314     - GAC ACA GAA GAC CAA GAA GAC CAA GTA GAT CC - #G CGG CTC ATT GAT GGG     8362     - AAG ATG ACC AGG CGG GGA GAC AGC CCC TGG CA - #G GTGGGAGGCG AGGCAGCACC     8415     - GGCTCGTCAC GTGCTGGGTC CGGGATCACT GAGTCCATCC TGGCAGCTAT GC - #TCAGGGTG     8475     - CAGAAACCGA GAGGGAAGCG CTGCCATTGC GTTTGGGGGA TGATGAAGGT GG - #GGGATGCT     8535     - TCAGGGAAAG ATGGACGCAA CCTGAGGGGA GAGGAGCAGC CAGGGTGGGT GA - #GGGGAGGG     8595     - GCATGGGGGC ATGGAGGGGT CTGCAGGAGG GAGGGTTACA GTTTCTAAAA AG - #AGCTGGAA     8655     - AGACACTGCT CTGCTGGCGG GATTTTAGGC AGAAGCCCTG CTGATGGGAG AG - #GGCTAGGA     8715     - GGGAGGGCCG GGCCTGAGTA CCCCTCCAGC CTCCACATGG GAACTGACAC TT - #ACTGGGTT     8775     - CCCCTCTCTG CCAGGCATGG GGGAGATAGG AACCAACAAG TGGGAGTATT TG - #CCCTGGGG     8835     - ACTCAGACTC TGCAAGGGTC AGGACCCCAA AGACCCGGCA GCCCAGTGGG AC - #CACAGCCA     8895     - GGACGGCCCT TCAAGATAGG GGCTGAGGGA GGCCAAGGGG AACATCCAGG CA - #GCCTGGGG     8955     - GCCACAAAGT CTTCCTGGAA GACACAAGGC CTGCCAAGCC TCTAAGGATG AG - #AGGAGCTC     9015     - GCTGGGCGAT GTTGGTGTGG CTGAGGGTGA CTGAAACAGT ATGAACAGTG CA - #GGAACAGC     9075     - ATGGGCAAAG GCAGGAAGAC ACCCTGGGAC AGGCTGACAC TGTAAAATGG GC - #AAAAATAG     9135     - AAAACGCCAG AAAGGCCTAA GCCTATGCCC ATATGACCAG GGAACCCAGG AA - #AGTGCATA     9195     - TGAAACCCAG GTGCCCTGGA CTGGAGGCTG TCAGGAGGCA GCCCTGTGAT GT - #CATCATCC     9255     - CACCCCATTC CAG GTG GTC CTG CTG GAC TCA AAG AA - #G AAG CTG GCC TGC     9304     - GGG GCA GTG CTC ATC CAC CCC TCC TGG GTG CT - #G ACA GCG GCC CAC TGC     9352     - ATG GAT GAG TCC AAG AAG CTC CTT GTC AGG CT - #T  G GTATGGGCTG     939 - #6     - GAGCCAGGCA GAAGGGGGCT GCCAGAGGCC TGGGTAGGGG GACCAGGCAG GC - #TGTTCAGG     9456     - TTTGGGGGAC CCCGCTCCCC AGGTGCTTAA GCAAGAGGCT TCTTGAGCTC CA - #CAGAAGGT     9516     - GTTTGGGGGG AAGAGGCCTA TGTGCCCCCA CCCTGCCCAC CCATGTACAC CC - #AGTATTTT     9576     - GCAGTAGGGG GTTCTCTGGT GCCCTCTTCG AATCTGGGCA CAGGTACCTG CA - #CACACATG     9636     - TTTGTGAGGG GCTACACAGA CCTTCACCTC TCCACTCCCA CTCATGAGGA GC - #AGGCTGTG     9696     - TGGGCCTCAG CACCCTTGGG TGCAGAGACC AGCAAGGCCT GGCCTCAGGG CT - #GTGCCTCC     9756     - CACAGACTGA CAGGGATGGA GCTGTACAGA GGGAGCCCTA GCATCTGCCA AA - #GCCACAAG     9816     - CTGCTTCCCT AGCAGGCTGG GGGCTCCTAT GCATTGGCCC CGATCTATGG CA - #ATTTCTGG     9876     - AGGGGGGGTC TGGCTCAACT CTTTATGCCA AAAAGAAGGC AAAGCATATT GA - #GAAAGGCC     9936     - AAATTCACAT TTCCTACAGC ATAATCTATG CCAGTGGCCC CGTGGGGCTT GG - #CTTAGAAT     9996     - TCCCAGGTGC TCTTCCCAGG GAACCATCAG TCTGGACTGA GAGGACCTTC TC - #TCTCAGGT     10056     - GGGACCCGGC CCTGTCCTCC CTGGCAGTGC CGTGTTCTGG GGGTCCTCCT CT - #CTGGGTCT     10116     - CACTGCCCCT GGGGTCTCTC CAGCTACCTT TGCTCCATGT TCCTTTGTGG CT - #CTGGTCTG     10176     - TGTCTGGGGT TTCCAGGGGT CTCGGGCTTC CCTGCTGCCC ATTCCTTCTC TG - #GTCTCACG     10236     - GCTCCGTGAC TCCTGAAAAC CAACCAGCAT CCTACCCCTT TGGATTGACA CC - #TGTTGGCC     10296     - ACTCCTTCTG GCAGGAAAAG TCACCGTTGA TAGGGTTCCA CGGCATAGAC AG - #GTGGCTCC     10356     - GCGCCAGTGC CTGGGACGTG TGGGTGCACA GTCTCCGGGT GAACCTTCTT CA - #GGCCCTCT     10416     - CCCAGGCCTG CAGGGGCACA GCAGTGGGTG GGCCTCAGGA AAGTGCCACT GG - #GGAGAGGC     10476     #TAT GAC CTG     10529C TGTGCCCTCT GCCCTGCAG  GA GAG     - CGG CGC TGG GAG AAG TGG GAG CTG GAC CTG GA - #C ATC AAG GAG GTC TTC     10577     - GTC CAC CCC AAC TAC AGC AAG AGC ACC ACC GA - #C AAT GAC ATC GCA CTG     10625     - CTG CAC CTG GCC CAG CCC GCC ACC CTC TCG CA - #G ACC ATA GTG CCC ATC     10673     - TGC CTC CCG GAC AGC GGC CTT GCA GAG CGC GA - #G CTC AAT CAG GCC GGC     10721     - CAG GAG ACC CTC GTG ACG GGC TGG GGC TAC CA - #C AGC AGC CGA GAG AAG     10769     - GAG GCC AAG AGA AAC CGC ACC TTC GTC CTC AA - #C TTC ATC AAG ATT CCC     10817     - GTG GTC CCG CAC AAT GAG TGC AGC GAG GTC AT - #G AGC AAC ATG GTG TCT     10865     - GAG AAC ATG CTG TGT GCG GGC ATC CTC GGG GA - #C CGG CAG GAT GCC TGC     10913     - GAG GGC GAC AGT GGG GGG CCC ATG GTC GCC TC - #C TTC CAC GGC ACC TGG     10961     - TTC CTG GTG GGC CTG GTG AGC TGG GGT GAG GG - #C TGT GGG CTC CTT CAC     11009     - AAC TAC GGC GTT TAC ACC AAA GTC AGC CGC TA - #C CTC GAC TGG ATC CAT     11057     - GGG CAC ATC AGA GAC AAG GAA GCC CCC CAG AA - #G AGC TGG GCA CCT     11102     - TAGCGACCCT CCCTGCAGGG CTGGGCTTTT GCATGGCAAT GGATGGGACA TT - #AAAGGGAC     11162     - ATGTAACAAG CACACCGGCC TGCTGTTCTG TCCTTCCATC CCTCTTTTGG GC - #TCTTCTGG     11222     - AGGGAAGTAA CATTTACTGA GCACCTGTTG TATGTCACAT GCCTTATGAA TA - #GAATCTTA     11282     - ACTCCTAGAG CAACTCTGTG GGGTGGGGAG GAGCAGATCC AAGTTTTGCG GG - #GTCTAAAG     11342     - CTGTGTGTGT TGAGGGGGAT ACTCTGTTTA TGAAAAAGAA TAAAAAACAC AA - #CCACGAAG     11402     - CCACTAGAGC CTTTTCCAGG GCTTTGGGAA GAGCCTGTGC AAGCCGGGGA TG - #CTGAAGGT     11462     - GAGGCTTGAC CAGCTTTCCA GCTAGCCCAG CTATGAGGTA GACATGTTTA GC - #TCATATCA     11522     - CAGAGGAGGA AACTGAGGGG TCTGAAAGGT TTACATGGTG GAGCCAGGAT TC - #AAATCTAG     11582     - GTCTGACTCC AAAACCCAGG TGCTTTTTTC TGTTCTCCAC TGTCCTGGAG GA - #CAGCTGTT     11642     - TCGACGGTGC TCAGTGTGGA GGCCACTATT AGCTCTGTAG GGAAGCAGCC AG - #AGACCCAG     11702     #             11725CCAG AAT     - (2) INFORMATION FOR SEQ ID NO:2:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 460 amino               (B) TYPE: amino acid               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:     - Met Trp Gln Leu Thr Ser Leu Leu Leu Phe Va - #l Ala Thr Trp Gly Ile     #                 15     - Ser Gly Thr Pro Ala Pro Leu Asp Ser Val Ph - #e Ser Ser Ser Glu Arg     #             30     - Ala His Gln Val Leu Arg Ile Arg Lys Arg Al - #a Asn Ser Phe Leu Glu     #         45     - Glu Leu Arg His Ser Ser Leu Glu Arg Glu Cy - #s Ile Glu Glu Ile Cys     #     60     - Asp Phe Glu Glu Ala Lys Glu Ile Phe Gln As - #n Val Asp Asp Thr Leu     # 80     - Ala Phe Trp Ser Lys His Val Asp Gly Asp Gl - #n Cys Leu Val Leu Pro     #                 95     - Leu Glu His Pro Cys Ala Ser Leu Cys Cys Gl - #y His Gly Thr Cys Ile     #           110     - Asp Gly Ile Gly Ser Phe Ser Cys Asp Cys Ar - #g Ser Gly Trp Glu Gly     #       125     - Arg Phe Cys Gln Arg Glu Val Ser Phe Leu As - #n Cys Ser Leu Asp Asn     #   140     - Gly Gly Cys Thr His Tyr Cys Leu Glu Glu Va - #l Gly Trp Arg Arg Cys     145                 1 - #50                 1 - #55                 1 -     #60     - Ser Cys Ala Pro Gly Tyr Lys Leu Gly Asp As - #p Leu Leu Gln Cys His     #               175     - Pro Ala Val Lys Phe Pro Cys Gly Arg Pro Tr - #p Lys Arg Met Glu Lys     #           190     - Lys Arg Ser His Leu Lys Arg Asp Thr Glu As - #p Gln Glu Asp Gln Val     #       205     - Asp Pro Arg Leu Ile Asp Gly Lys Met Thr Ar - #g Arg Gly Asp Ser Pro     #   220     - Trp Gln Val Val Leu Leu Asp Ser Lys Lys Ly - #s Leu Ala Cys Gly Ala     225                 2 - #30                 2 - #35                 2 -     #40     - Val Leu Ile His Pro Ser Trp Val Leu Thr Al - #a Ala His Cys Met Asp     #               255     - Glu Ser Lys Lys Leu Leu Val Arg Leu Gly Gl - #u Tyr Asp Leu Arg Arg     #           270     - Trp Glu Lys Trp Glu Leu Asp Leu Asp Ile Ly - #s Glu Val Phe Val His     #       285     - Pro Asn Tyr Ser Lys Ser Thr Thr Asp Asn As - #p Ile Ala Leu Leu His     #   300     - Leu Ala Gln Pro Ala Thr Leu Ser Gln Thr Il - #e Val Pro Ile Cys Leu     305                 3 - #10                 3 - #15                 3 -     #20     - Pro Asp Ser Gly Leu Ala Glu Arg Glu Leu As - #n Gln Ala Gly Gln Glu     #               335     - Thr Leu Val Thr Gly Trp Gly Tyr His Ser Se - #r Arg Glu Lys Glu Ala     #           350     - Lys Arg Asn Arg Thr Phe Val Leu Asn Phe Il - #e Lys Ile Pro Val Val     #       365     - Pro His Asn Glu Cys Ser Glu Val Met Ser As - #n Met Val Ser Glu Asn     #   380     - Met Leu Cys Ala Gly Ile Leu Gly Asp Arg Gl - #n Asp Ala Cys Glu Gly     385                 3 - #90                 3 - #95                 4 -     #00     - Asp Ser Gly Gly Pro Met Val Ala Ser Phe Hi - #s Gly Thr Trp Phe Leu     #               415     - Val Gly Leu Val Ser Trp Gly Glu Gly Cys Gl - #y Leu Leu His Asn Tyr     #           430     - Gly Val Tyr Thr Lys Val Ser Arg Tyr Leu As - #p Trp Ile His Gly His     #       445     - Ile Arg Asp Lys Glu Ala Pro Gln Lys Ser Tr - #p Ala     #   460     - (2) INFORMATION FOR SEQ ID NO:3:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 1386 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: double               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     -     (ix) FEATURE:               (A) NAME/KEY: CDS               (B) LOCATION: 1..1380     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:     - ATG TGG CAG CTC ACA AGC CTC CTG CTG TTC GT - #G GCC ACC TGG GGA ATT       48     Met Trp Gln Leu Thr Ser Leu Leu Leu Phe Va - #l Ala Thr Trp Gly Ile     #                 15     - TCC GGC ACA CCA GCT CCT CTT GAC TCA GTG TT - #C TCC AGC AGC GAG CGT       96     Ser Gly Thr Pro Ala Pro Leu Asp Ser Val Ph - #e Ser Ser Ser Glu Arg     #             30     - GCC CAC CAG GTG CTG CGG ATC CGC AAA CGT GC - #C AAC TCC TTC CTG GAG      144     Ala His Gln Val Leu Arg Ile Arg Lys Arg Al - #a Asn Ser Phe Leu Glu     #         45     - GAG CTC CGT CAC AGC AGC CTG GAG CGG GAG TG - #C ATA GAG GAG ATC TGT      192     Glu Leu Arg His Ser Ser Leu Glu Arg Glu Cy - #s Ile Glu Glu Ile Cys     #     60     - GAC TTC GAG GAG GCC AAG GAA ATT TTC CAA AA - #T GTG GAT GAC ACA CTG      240     Asp Phe Glu Glu Ala Lys Glu Ile Phe Gln As - #n Val Asp Asp Thr Leu     # 80     - GCC TTC TGG TCC AAG CAC GTC GAC GGT GAC CA - #G TGC TTG GTC TTG CCC      288     Ala Phe Trp Ser Lys His Val Asp Gly Asp Gl - #n Cys Leu Val Leu Pro     #                 95     - TTG GAG CAC CCG TGC GCC AGC CTG TGC TGC GG - #G CAC GGC ACG TGC ATC      336     Leu Glu His Pro Cys Ala Ser Leu Cys Cys Gl - #y His Gly Thr Cys Ile     #           110     - GAC GGC ATC GGC AGC TTC AGC TGC GAC TGC CG - #C AGC GGC TGG GAG GGC      384     Asp Gly Ile Gly Ser Phe Ser Cys Asp Cys Ar - #g Ser Gly Trp Glu Gly     #       125     - CGC TTC TGC CAG CGC GAG GTG AGC TTC CTC AA - #T TGC TCT CTG GAC AAC      432     Arg Phe Cys Gln Arg Glu Val Ser Phe Leu As - #n Cys Ser Leu Asp Asn     #   140     - GGC GGC TGC ACG CAT TAC TGC CTA GAG GAG GT - #G GGC TGG CGG CGC TGT      480     Gly Gly Cys Thr His Tyr Cys Leu Glu Glu Va - #l Gly Trp Arg Arg Cys     145                 1 - #50                 1 - #55                 1 -     #60     - AGC TGT GCG CCT GGC TAC AAG CTG GGG GAC GA - #C CTC CTG CAG TGT CAC      528     Ser Cys Ala Pro Gly Tyr Lys Leu Gly Asp As - #p Leu Leu Gln Cys His     #               175     - CCC GCA GTG AAG TTC CCT TGT GGG AGG CCC TG - #G AAG CGG ATG GAG AAG      576     Pro Ala Val Lys Phe Pro Cys Gly Arg Pro Tr - #p Lys Arg Met Glu Lys     #           190     - AAG CGC AGT CAC CTG AAA CGA GAC ACA GAA GA - #C CAA GAA GAC CAA GTA      624     Lys Arg Ser His Leu Lys Arg Asp Thr Glu As - #p Gln Glu Asp Gln Val     #       205     - GAT CCG CGG CTC ATT GAT GGG AAG ATG ACC AG - #G CGG GGA GAC AGC CCC      672     Asp Pro Arg Leu Ile Asp Gly Lys Met Thr Ar - #g Arg Gly Asp Ser Pro     #   220     - TGG CAG GTG GTC CTG CTG GAC TCA AAG AAG AA - #G CTG GCC TGC GGG GCA      720     Trp Gln Val Val Leu Leu Asp Ser Lys Lys Ly - #s Leu Ala Cys Gly Ala     225                 2 - #30                 2 - #35                 2 -     #40     - GTG CTC ATC CAC CCC TCC TGG GTG CTG ACA GC - #G GCC CAC TGC ATG GAT      768     Val Leu Ile His Pro Ser Trp Val Leu Thr Al - #a Ala His Cys Met Asp     #               255     - GAG TCC AAG AAG CTC CTT GTC AGG CTT GGA GA - #G TAT GAC CTG CGG CGC      816     Glu Ser Lys Lys Leu Leu Val Arg Leu Gly Gl - #u Tyr Asp Leu Arg Arg     #           270     - TGG GAG AAG TGG GAG CTG GAC CTG GAC ATC AA - #G GAG GTC TTC GTC CAC      864     Trp Glu Lys Trp Glu Leu Asp Leu Asp Ile Ly - #s Glu Val Phe Val His     #       285     - CCC AAC TAC AGC AAG AGC ACC ACC GAC AAT GA - #C ATC GCA CTG CTG CAC      912     Pro Asn Tyr Ser Lys Ser Thr Thr Asp Asn As - #p Ile Ala Leu Leu His     #   300     - CTG GCC CAG CCC GCC ACC CTC TCG CAG ACC AT - #A GTG CCC ATC TGC CTC      960     Leu Ala Gln Pro Ala Thr Leu Ser Gln Thr Il - #e Val Pro Ile Cys Leu     305                 3 - #10                 3 - #15                 3 -     #20     - CCG GAC AGC GGC CTT GCA GAG CGC GAG CTC AA - #T CAG GCC GGC CAG GAG     1008     Pro Asp Ser Gly Leu Ala Glu Arg Glu Leu As - #n Gln Ala Gly Gln Glu     #               335     - ACC CTC GTG ACG GGC TGG GGC TAC CAC AGC AG - #C CGA GAG AAG GAG GCC     1056     Thr Leu Val Thr Gly Trp Gly Tyr His Ser Se - #r Arg Glu Lys Glu Ala     #           350     - AAG AGA AAC CGC ACC TTC GTC CTC AAC TTC AT - #C AAG ATT CCC GTG GTC     1104     Lys Arg Asn Arg Thr Phe Val Leu Asn Phe Il - #e Lys Ile Pro Val Val     #       365     - CCG CAC AAT GAG TGC AGC GAG GTC ATG AGC AA - #C ATG GTG TCT GAG AAC     1152     Pro His Asn Glu Cys Ser Glu Val Met Ser As - #n Met Val Ser Glu Asn     #   380     - ATG CTG TGT GCG GGC ATC CTC GGG GAC CGG CA - #G GAT GCC TGC GAG GGC     1200     Met Leu Cys Ala Gly Ile Leu Gly Asp Arg Gl - #n Asp Ala Cys Glu Gly     385                 3 - #90                 3 - #95                 4 -     #00     - GAC AGT GGG GGG CCC ATG GTC GCC TCC TTC CA - #C GGC ACC TGG TTC CTG     1248     Asp Ser Gly Gly Pro Met Val Ala Ser Phe Hi - #s Gly Thr Trp Phe Leu     #               415     - GTG GGC CTG GTG AGC TGG GGT GAG GGC TGT GG - #G CTC CTT CAC AAC TAC     1296     Val Gly Leu Val Ser Trp Gly Glu Gly Cys Gl - #y Leu Leu His Asn Tyr     #           430     - GGC GTT TAC ACC AAA GTC AGC CGC TAC CTC GA - #C TGG ATC CAT GGG CAC     1344     Gly Val Tyr Thr Lys Val Ser Arg Tyr Leu As - #p Trp Ile His Gly His     #       445     - ATC AGA GAC AAG GAA GCC CCC CAG AAG AGC TG - #G GCA CCTTAG     #1386     Ile Arg Asp Lys Glu Ala Pro Gln Lys Ser Tr - #p Ala     #   460     - (2) INFORMATION FOR SEQ ID NO:4:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 460 amino               (B) TYPE: amino acid               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:     - Met Trp Gln Leu Thr Ser Leu Leu Leu Phe Va - #l Ala Thr Trp Gly Ile     #                 15     - Ser Gly Thr Pro Ala Pro Leu Asp Ser Val Ph - #e Ser Ser Ser Glu Arg     #             30     - Ala His Gln Val Leu Arg Ile Arg Lys Arg Al - #a Asn Ser Phe Leu Glu     #         45     - Glu Leu Arg His Ser Ser Leu Glu Arg Glu Cy - #s Ile Glu Glu Ile Cys     #     60     - Asp Phe Glu Glu Ala Lys Glu Ile Phe Gln As - #n Val Asp Asp Thr Leu     # 80     - Ala Phe Trp Ser Lys His Val Asp Gly Asp Gl - #n Cys Leu Val Leu Pro     #                 95     - Leu Glu His Pro Cys Ala Ser Leu Cys Cys Gl - #y His Gly Thr Cys Ile     #           110     - Asp Gly Ile Gly Ser Phe Ser Cys Asp Cys Ar - #g Ser Gly Trp Glu Gly     #       125     - Arg Phe Cys Gln Arg Glu Val Ser Phe Leu As - #n Cys Ser Leu Asp Asn     #   140     - Gly Gly Cys Thr His Tyr Cys Leu Glu Glu Va - #l Gly Trp Arg Arg Cys     145                 1 - #50                 1 - #55                 1 -     #60     - Ser Cys Ala Pro Gly Tyr Lys Leu Gly Asp As - #p Leu Leu Gln Cys His     #               175     - Pro Ala Val Lys Phe Pro Cys Gly Arg Pro Tr - #p Lys Arg Met Glu Lys     #           190     - Lys Arg Ser His Leu Lys Arg Asp Thr Glu As - #p Gln Glu Asp Gln Val     #       205     - Asp Pro Arg Leu Ile Asp Gly Lys Met Thr Ar - #g Arg Gly Asp Ser Pro     #   220     - Trp Gln Val Val Leu Leu Asp Ser Lys Lys Ly - #s Leu Ala Cys Gly Ala     225                 2 - #30                 2 - #35                 2 -     #40     - Val Leu Ile His Pro Ser Trp Val Leu Thr Al - #a Ala His Cys Met Asp     #               255     - Glu Ser Lys Lys Leu Leu Val Arg Leu Gly Gl - #u Tyr Asp Leu Arg Arg     #           270     - Trp Glu Lys Trp Glu Leu Asp Leu Asp Ile Ly - #s Glu Val Phe Val His     #       285     - Pro Asn Tyr Ser Lys Ser Thr Thr Asp Asn As - #p Ile Ala Leu Leu His     #   300     - Leu Ala Gln Pro Ala Thr Leu Ser Gln Thr Il - #e Val Pro Ile Cys Leu     305                 3 - #10                 3 - #15                 3 -     #20     - Pro Asp Ser Gly Leu Ala Glu Arg Glu Leu As - #n Gln Ala Gly Gln Glu     #               335     - Thr Leu Val Thr Gly Trp Gly Tyr His Ser Se - #r Arg Glu Lys Glu Ala     #           350     - Lys Arg Asn Arg Thr Phe Val Leu Asn Phe Il - #e Lys Ile Pro Val Val     #       365     - Pro His Asn Glu Cys Ser Glu Val Met Ser As - #n Met Val Ser Glu Asn     #   380     - Met Leu Cys Ala Gly Ile Leu Gly Asp Arg Gl - #n Asp Ala Cys Glu Gly     385                 3 - #90                 3 - #95                 4 -     #00     - Asp Ser Gly Gly Pro Met Val Ala Ser Phe Hi - #s Gly Thr Trp Phe Leu     #               415     - Val Gly Leu Val Ser Trp Gly Glu Gly Cys Gl - #y Leu Leu His Asn Tyr     #           430     - Gly Val Tyr Thr Lys Val Ser Arg Tyr Leu As - #p Trp Ile His Gly His     #       445     - Ile Arg Asp Lys Glu Ala Pro Gln Lys Ser Tr - #p Ala     #   460     - (2) INFORMATION FOR SEQ ID NO:5:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 10807 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:     - ACGCGTGTCG ACCTGCAGGT CAACGGATCT CTGTGTCTGT TTTCATGTTA GT - #ACCACACT       60     - GTTTTGGTGG CTGTAGCTTT CAGCTACAGT CTGAAGTCAT AAAGCCTGGT AC - #CTCCAGCT      120     - CTGTTCTCTC TCAAGATTGT GTTCTGCTGT TTGGGTCTTT AGTGTCTCCA CA - #CAATTTTT      180     - AGAATTGTTT GTTCTAGTTC TGTGAAAAAT GATGCTGGTA TTTTGATAAG GA - #TTGCATTG      240     - AATCTGTAAA GCTACAGATA TAGTCATTGG GTAGTACAGT CACTTTAACA AT - #ATTAACTC      300     - TTCACATCTG TGAGCATGAT ATATTTTCCC CCTCTATATC ATCTTCAATT CC - #TCCTATCA      360     - GTTTCTTTCA TTGCAGTTTT CTGAGTACAG GTCTTACACC TCCTTGGTTA GA - #GTCATTCC      420     - TCAGTATTTT ATTCCTTTGA TACAATTGTG AATGAGGTAA TTTTCTTAGT TT - #CTCTTTCT      480     - GATAGCTCAT TGTTAGTGTA TATATAGAAA AGCAACAGAT TTCTATGTAT TA - #ATTTTGTA      540     - TCCTGCAACA GATTTCTATG TATTAATTTT GTATCCTGCT ACTTTACGGA AT - #TCACTTAT      600     - TAGCTTTTTG GTGACATCTT GAGGATTTTC TGAAGAAAAT GGCATGGTAT GG - #TAGGACAA      660     - GGTGTCATGT CATCTGCAAA CAGTGGCAGT TTTCCTTCTT CCCTTCCAAC CT - #GGATTTCT      720     - TTGATTTCTT TCTGTCTGAG TACGACTAGG ATTCCCAATA CTATACCGAA TA - #AAAGTGGC      780     - AAGAGTGGAC ATCCTTGTCT TATTTTTCTG ACCTTAGAGG AAATGCTTTC AG - #TTTTTCAC      840     - CATTAATTAT AATGTTTACT GTGGGCTTGT CATATGTGGC CTTCATTATA TG - #GAGGTCTA      900     - TTCCCTCTAT ACCCACCTTG TTGAGAGTTT TTATCATAAA AGTATGTTGA AT - #TTTGTCAA      960     - AAGTTTTTCC TGCATCTATT GAGATGATTT TTACTCTTCA ATTCATTAAT GA - #TTTTTATT     1020     - CTTCATTTTG TTAATGATTT CCATTCTTCA ATTTGTTAAC GTGGTATATC AC - #ATTGATTG     1080     - ATTTGTGGAT ACCTTTGTAT CCCTGGGATA AACCTCACTT GATCATGAGC TT - #TCAATGTA     1140     - TTTTTGAATT CACTTTGCTA ATATTCTGTT GGGTATTTTT GCATCTCTAT TC - #ATCAATGA     1200     - TATTGGCCTA AGAAAGGTTT TGTCTGGTTT TAGTATCAGG GTGATGCTGG CC - #TCATAGAG     1260     - AGAGTTTAGA AGCATTTCCT CCTCTTTGAT TTTTCGGAAT AGTTTGAGTA GG - #ATAGGTAT     1320     - TAACTCTTCT TTAAATGTTT GGGGACTTCC CTGGTGAGCC GGTGGTTGAG AA - #TCCGCCTC     1380     - AGGGATGTGG GTTTGATCCC TGGTCAGGGA ACCATTAATA AGATCCCACA TG - #CTGCAGGC     1440     - AACAAGCCCC CAAGCTGCAA CCACTGAGCT GCAACCGCTG CAGTGCCCAC AG - #GCCACGAC     1500     - CAGAGAAAGC CCACATACAG CAGGGAAGAC CCAGCACAAC CGGAAAAAGG AG - #TTTGGTGG     1560     - AATACAGCTG TGAAGCCGTC TGGTCCTGGA CTCCTGCTTG AGGGAATTTT TT - #AAAAATTA     1620     - TTGATTCAAT TTCATTACTG GTAACTGGTC TGTTCATATT TTCTATTTCT TC - #CGGGTTCA     1680     - GTCTTGGGAG ATTGTACATG CCTAGGAATG TGTCCGTTTC TTCTAGGTTG TC - #CATTTTAT     1740     - TGGACATGCA TGGGAGCACA CAGCACCGAC CAGCGAGACT CATGCTGGCT TC - #CTGGGGCC     1800     - AGGCTGGGGC CCCAAGCAGC ATGGCATCCT AGAGTGTGTG AAAGCCCACT GA - #CCCTGCCC     1860     - AGCCCCACAA TTTCATTCTG AGAAGTGATT CCTTGCTTCT GCACTTACAG GC - #CCAGGATC     1920     - TGACCTGCTT CTGAGGAGCA GGGGTTTTGG CAGGACGGGG AGATGCTGAG AG - #CCGACGGG     1980     - GGTCCAGGTC CCCTCCCAGG CCCCCCTGTC TGGGGCAGCC CTTGGGAAAG AT - #TGCCCCAG     2040     - TCTCCCTCCT ACAGTGGTCA GTCCCAGCTG CCCCAGGCCA GAGCTGCTTT AT - #TTCCGTCT     2100     - CTCTCTCTGG ATGGTATTCT CTGGAAGCTG AAGGTTCCTG AAGTTATGAA TA - #GCTTTGCC     2160     - CTGAAGGGCA TGGTTTGTGG TCACGGTTCA CAGGAACTTG GGAGACCCTG CA - #GCTCAGAC     2220     - GTCCCGAGAT TGGTGGCACC CAGATTTCCT AAGCTCGCTG GGGAACAGGG CG - #CTTGTTTC     2280     - TCCCTGGCTG ACCTCCCTCC TCCCTGCATC ACCCAGTTCT GAAAGCAGAG CG - #GTGCTGGG     2340     - GTCACAGCCT CTCGCATCTA ACGCCGGTGT CCAAACCACC CGTGCTGGTG TT - #CGGGGGGC     2400     - TACCTATGGG GAAGGGCTTC TCACTGCAGT GGTGCCCCCC GTCCCCTCTG AG - #ATCAGAAG     2460     - TCCCAGTCCG GACGTCAAAC AGGCCGAGCT CCCTCCAGAG GCTCCAGGGA GG - #GATCCTTG     2520     - CCCCCCCGCT GCTGCCTCCA GCTCCTGGTG CCGCACCCTT GAGCCTGATC TT - #GTAGACGC     2580     - CTCAGTCTAG TCTCTGCCTC CGTGTTCACA CGCCTTCTCC CCATGTCCCC TC - #CGTGTCCC     2640     - CGTTTTCTCT CACAAGGACA CCGGACATTA GATTAGCCCC TGTTCCAGCC TC - #ACCTGAAC     2700     - AGCTCACATC TGTAAAGACC TAGATTCCAA ACAAGATTCC AACCTGAAGT TC - #CCGGTGGA     2760     - TGTGAGTTCT GGGGCGACAT CCTTCAACCC CATCACAGCT TGCAGTTCAT CG - #CAAAACAT     2820     - GGAACCTGGG GTTTATCGTA AAACCCAGGT TCTTCATGAA ACACTGAGCT TC - #GAGGCTTG     2880     - TTGCAAGAAT TAAAGGTGCT AATACAGATC AGGGCAAGGA CTGAAGCTGG CT - #AAGCCTCC     2940     - TCTTTCCATC ACAGGAAAGG GGGGCCTGGG GGCGGCTGGA GGTCTGCTCC CG - #TGAGTGAG     3000     - CTCTTTCCTG CTACAGTCAC CAACAGTCTC TCTGGGAAGG AAACCAGAGG CC - #AGAGAGCA     3060     - AGCCGGAGCT AGTTTAGGAG ACCCCTGAAC CTCCACCCAA GATGCTGACC AG - #CCAGCGGG     3120     - CCCCCTGGAA AGACCCTACA GTTCAGGGGG GAAGAGGGGC TGACCCGCCA GG - #TCCCTGCT     3180     - ATCAGGAGAC ATCCCCGCTA TCAGGAGATT CCCCCACCTT GCTCCCGTTC CC - #CTATCCCA     3240     - ATACGCCCAC CCCACCCCTG TGATGAGCAG TTTAGTCACT TAGAATGTCA AC - #TGAAGGCT     3300     - TTTGCATCCC CTTTGCCAGA GGCACAAGGC ACCCACAGCC TGCTGGGTAC CG - #ACGCCCAT     3360     - GTGGATTCAG CCAGGAGGCC TGTCCTGCAC CCTCCCTGCT CGGGCCCCCT CT - #GTGCTCAG     3420     - CAACACACCC AGCACCAGCA TTCCCGCTGC TCCTGAGGTC TGCAGGCAGC TC - #GCTGTAGC     3480     - CTGAGCGGTG TGGAGGGAAG TGTCCTGGGA GATTTAAAAT GTGAGAGGCG GG - #AGGTGGGA     3540     - GGTTGGGCCC TGTGGGCCTG CCCATCCCAC GTGCCTGCAT TAGCCCCAGT GC - #TGCTCAGC     3600     - CGTGCCCCCG CCGCAGGGGT CAGGTCACTT TCCCGTCCTG GGGTTATTAT GA - #CTCTTGTC     3660     - ATTGCCATTG CCATTTTTGC TACCCTAACT GGGCAGCAGG TGCTTGCAGA GC - #CCTCGATA     3720     - CCGACCAGGT CCTCCCTCGG AGCTCGACCT GAACCCCATG TCACCCTTGC CC - #CAGCCTGC     3780     - AGAGGGTGGG TGACTGCAGA GATCCCTTCA CCCAAGGCCA CGGTCACATG GT - #TTGGAGGA     3840     - GCTGGTGCCC AAGGCAGAGG CCACCCTCCA GGACACACCT GTCCCCAGTG CT - #GGCTCTGA     3900     - CCTGTCCTTG TCTAAGAGGC TGACCCCGGA AGTGTTCCTG GCACTGGCAG CC - #AGCCTGGA     3960     - CCCAGAGTCC AGACACCCAC CTGTGCCCCC GCTTCTGGGG TCTACCAGGA AC - #CGTCTAGG     4020     - CCCAGAGGGG ACTTCCTGCT TGGCCTTGGA TGGAAGAAGG CCTCCTATTG TC - #CTCGTAGA     4080     - GGAAGCCACC CCGGGGCCTG AGGATGAGCC AAGTGGGATT CCGGGAACCG CG - #TGGCTGGG     4140     - GGCCCAGCCC GGGCTGGCTG GCCTGCATGC CTCCTGTATA AGGCCCCAAG CC - #TGCTGTCT     4200     - CAGCCCTCCA CTCCCTGCAG AGCTCAGAAG CACGACCCCA GGGATATCCC TG - #CAGCCATG     4260     - AAGTGCCTCC TGCTTGCCCT GGGCCTGGCC CTCGCCTGTG GCGTCCAGGC CA - #TCATCGTC     4320     - ACCCAGACCA TGAAAGGCCT GGACATCCAG AAGGTTCGAG GGTTGGCCGG GT - #GGGTGAGT     4380     - TGCAGGGCGG GCAGGGGAGC TGGGCCTCAG AGAGCCAAGA GAGGCTGTGA CG - #TTGGGTTC     4440     - CCATCAGTCA GCTAGGGCCA CCTGACAAAT CCCCGCTGGG GCAGCTTCAA CC - #AGGCGTTC     4500     - ACTGTCTTGC ATTCTGGAGG CTGGAAGCCC AAGATCCAGG TGTTGGCAGG GC - #TGGCTTCT     4560     - CCTGCGGCCG CTCTCTGGGG AGCAGACGGC CGTCTTCTCC AGTCCTCTGC GC - #GCCCTGAT     4620     - TTCCTCTTCC TGTGAGGCCA CCAGGCCTGC TGGAAACACG CCTGCCTGCG CA - #GCTTCACA     4680     - CGACCTTTGT CATCTCTTTA AAGGCCATGT CTCCAGAGTC ATGTGTTGAA GT - #TCTGGGGG     4740     - TTAGTGGGAC ACAGTTCAGC CCCTAAAAGA GTCTCTCTGC CCCTCAAATT TT - #CCCCACCT     4800     - CCAGCCATGT CTCCCCAAGA TCCAAATGTT GCTACATGTG GGGGGGCTCA TC - #TGGGTCCC     4860     - TCTTTGGGTT CAGTGTGAGT CTGGGGAGAG CATTCCCCAG GGTGCAGAGT TG - #GGGGGAGT     4920     - ATCTCAGGGC TGCCCAGGCC GGGGTGGGAC AGAGAGCCCA CTGTGGGGCT GG - #GGGCCCCT     4980     - TCCCACCCCC AGAGTGCAAC TCAAGGTCCC TCTCCAGGTG GCGGGGACTT GG - #CACTCCTT     5040     - GGCTATGGCG GCCAGCGACA TCTCCCTGCT GGATGCCCAG AGTGCCCCCC TG - #AGAGTGTA     5100     - CGTGGAGGAG CTGAAGCCCA CCCCCGAGGG CAACCTGGAG ATCCTGCTGC AG - #AAATGGTG     5160     - GGCGTCTCTC CCCAACATGG AACCCCCACT CCCCAGGGCT GTGGACCCCC CG - #GGGGGTGG     5220     - GGTGCAGGAG GGACCAGGGC CCCAGGGCTG GGGAAGAGGG CTCAGAGTTT AC - #TGGTACCC     5280     - GGCGCTCCAC CCAAGGCTGC CCACCCAGGG CTTTTTTTTT TTTTAAACTT TT - #ATTAATTT     5340     - GATGCTTCAG AACATCATCA AACAAATGAA CATAAAACAT TCATTTTTGT TT - #ACTTGGAA     5400     - GGGGAGATAA AATCCTCTGA AGTGGAAATG CATAGCAAAG ATACATACAA TG - #AGGCAGGT     5460     - ATTCTGAATT CCCTGTTAGT CTGAGGATTA CAAGTGTATT TGAGCAACAG AG - #AGACATTT     5520     - TCATCATTTC TAGTCTGAAC ACCTCAGTAT CTAAAATGAA CAAGAAGTCC TG - #GAAACGAA     5580     - GCAGTGTGGG GATAGGCCCG TGTGAAGGCT GCTGGGAGGC AGCAGACCTG GG - #TCTTCGGG     5640     - CTCAAGCAGT TCCCGCTACC AGCCCTGTCC ACCTCAGACG GGGGTCAGGG TG - #CAGGAGAG     5700     - AGCTGGATGG GTGTGGGGGC AGAGATGGGG ACCTGAACCC CAGGGCTGCC TT - #TTGGGGGT     5760     - GCCTGTGGTC AAGGCTCTCC CTGACCTTTT CTCTCTGGCT TCATCTGACT TC - #TCCTGGCC     5820     - CATCCACCCG GTCCCCTGTG GCCTGAGGTG ACAGTGAGTG CGCCGAGGCT AG - #TTGGCCAG     5880     - CTGGCTCCTA TGCCCATGCC ACCCCCCTCC AGCCCTCCTG GGCCAGCTTC TG - #CCCCTGGC     5940     - CCTCAGTTCA TCCTGATGAA AATGGTCCAT GCCAATGGCT CAGAAAGCAG CT - #GTCTTTCA     6000     - GGGAGAACGG CGAGTGTGCT CAGAAGAAGA TTATTGCAGA AAAAACCAAG AT - #CCCTGCGG     6060     - TGTTCAAGAT CGATGGTGAG TCCGGGTCCC TGGGGGACAC CCACCACCCC CG - #CCCCCGGG     6120     - GACTGTGGAC AGGTTCAGGG GGCTGGCGTC GGGCCCTGGG ATGCTAAGGG AC - #TGGTGGTG     6180     - ATGAAGACAC TGCCTTGACA CCTGCTTCAC TTGCCTCCCC TGCCACCTGC CC - #GGGGCCTT     6240     - GGGGCGGTGG CCATGGGCAG GTCCCGGCTG GCGGGCTAAC CCACCAGGGT GA - #CACCCGAG     6300     - CTCTCTTTGC TGGGGGGCGG GCGGTGCTCT GGGCCCTCAG GCTGAGCTCA GG - #AGGTACCT     6360     - GTGCCCTCCC AGGGGTAACC GAGAGCCGTT GCCCACTCCA GGGGCCCAGG TG - #CCCCACGA     6420     - CCCCAGCCCG CTCCACAGCT CCTTCATCTC CTGGAGACAA ACTCTGTCCG CC - #CTCGCTCA     6480     - TTCACTTGTT CGTCCTAAAT CCGAGATGAT AAAGCTTCGA GGGGGGGTTG GG - #GTTCCATC     6540     - AGGGCTGCCC TTCCGCCGGG CAGCCTGGGC CACATCTGCC CTTGGCCCCC TC - #AGGACTCA     6600     - CTCTGACTGG AGGCCCTGCA CTGACTGACG CCAGGGTGCC CAGCCCAGGG TC - #TCTGGCGC     6660     - CATCCAGCTG CACTGGGTTT GGGTGCTGGT CCTGCCCCCA AGCTGCCCGG AC - #ACCACAGG     6720     - CAGCCGGGGC TGCCCACTGG CCTCGGTCAG GGTGAGCCCC AGCTGCCCCC GC - #TCAGGGCT     6780     - TGCCCCGACA ATGACCCCAT CCTCAGGACG CACCCCCCTT CCCTTGCTGG GC - #AGTGTCCA     6840     - GCCCCACCCG AGATCGGGGG AAGCCCTATT TCTTGACAAC TCCAGTCCCT GG - #GGGAGGGG     6900     - GCCTCAGACT GAGTGGTGAG TGTTCCCAAG TCCAGGAGGT GGTGGAGGGT CC - #TGGCGGAT     6960     - CCAGAGTTGA CAGTGAGGGC TTCCTGGGCC CCATGCGCCT GGCAGTGGCA GC - #AGGGAAGA     7020     - GGAAGCACCA TTTCAGGGGT GGGGGATGCC AGAGGCGCTC CCCACCCCGT CT - #TCGCCGGG     7080     - TGGTGACCCC GGGGGAGCCC CGCTGGTCGT GGAGGGTGCT GGGGGCTGAC TA - #GCAACCCC     7140     - TCCCCCCCCG TTGGAACTCA CTTTTCTCCC GTCTTGACCG CGTCCAGCCT TG - #AATGAGAA     7200     - CAAAGTCCTT GTGCTGGACA CCGACTACAA AAAGTACCTG CTCTTCTGCA TG - #GAAAACAG     7260     - TGCTGAGCCC GAGCAAAGCC TGGCCTGCCA GTGCCTGGGT GGGTGCCAAC CC - #TGGCTGCC     7320     - CAGGGAGACC AGCTGCGTGG TCCTTGCTGC AACAGGGGGT GGGGGGTGGG AG - #CTTGATCC     7380     - CCAGGAGGAG GAGGGGTGGG GGGTCCCTGA GTCCCGCCAG GAGAGAGTGG TC - #GCATACCG     7440     - GGAGCCAGTC TGCTGTGGGC CTGTGGGTGG CTGGGGACGG GGGCCAGACA CA - #CAGGCCGG     7500     - GAGACGGGTG GGCTGCAGAA CTGTGACTGG TGTGACCGTC GCGATGGGGC CG - #GTGGTCAC     7560     - TGAATCTAAC AGCCTTTGTT ACCGGGGAGT TTCAATTATT TCCCAAAATA AG - #AACTCAGG     7620     - TACAAAGCCA TCTTTCAACT ATCACATCCT GAAAACAAAT GGCAGGTGAC AT - #TTTCTGTG     7680     - CCGTAGCAGT CCCACTGGGC ATTTTCAGGG CCCCTGTGCC AGGGGGGCGC GG - #GCATCGGC     7740     - GAGTGGAGGC TCCTGGCTGT GTCAGCCGGC CCAGGGGGAG GAAGGGACCC GG - #ACAGCCAG     7800     - AGGTGGGGGG CAGGCTTTCC CCCTGTGACC TGCAGACCCA CTGCACTGCC CT - #GGGAGGAA     7860     - GGGAGGGGAA CTAGGCCAAG GGGGAAGGGC AGGTGCTCTG GAGGGCAAGG GC - #AGACCTGC     7920     - AGACCACCCT GGGGAGCAGG GACTGACCCC CGTCCCTGCC CCATAGTCAG GA - #CCCCGGAG     7980     - GTGGACAACG AGGCCCTGGA GAAATTCGAC AAAGCCCTCA AGGCCCTGCC CA - #TGCACATC     8040     - CGGCTTGCCT TCAACCCGAC CCAGCTGGAG GGTGAGCACC CAGGCCCCGC CC - #TTCCCCAG     8100     - GGCAGGAGCC ACCCGGCCCC GGGACGACCT CCTCCCATGG TGACCCCCAG CT - #CCCCAGGC     8160     - CTCCCAGGAG GAAGGGGTGG GGTGCAGCAC CCCGTGGGGG CCCCCTCCCC AC - #CCCCTGCC     8220     - AGGCCTCTCT TCCCGAGGTG TCCAGTCCCA TCCTGACCCC CCCATGACTC TC - #CCTCCCCC     8280     - ACAGGGCAGT GCCACGTCTA GGTGAGCCCC TGCCGGTGCC TCTGGGGTAA GC - #TGCCTGCC     8340     - CTGCCCCACG TCCTGGGCAC ACACATGGGG TAGGGGGTCT TGGTGGGGCC TG - #GGACCCCA     8400     - CATCAGGCCC TGGGGTCCCC CCTGTGAGAA TGGCTGGAAG CTGGGGTCCC TC - #CTGGCGAC     8460     - TGCAGAGCTG GCTGGCCGCG TGCCACTCTT GTGGGTGACC TGTGTCCTGG CC - #TCACACAC     8520     - TGACCTCCTC CAGCTCCTTC CAGCAGAGCT AAGGCTAAGT GAGCCAGAAT GG - #TACCTAAG     8580     - GGGAGGCTAG CGGTCCTTCT CCCGAGGAGG GGCTGTCCTG GAACCACCAG CC - #ATGGAGAG     8640     - GCTGGCAAGG GTCTGGCAGG TGCCCCAGGA ATCACAGGGG GGCCCCATGT CC - #ATTTCAGG     8700     - GCCCGGGAGC CTTGGACTCC TCTGGGGACA GACGACGTCA CCACCGCCCC CC - #CCCCATCA     8760     - GGGGGACTAG AAGGGACCAG GACTGCAGTC ACCCTTCCTG GGACCCAGGC CC - #CTCCAGGC     8820     - CCCTCCTGGG GCTCCTGCTC TGGGCAGCTT CTCCTTCACC AATAAAGGCA TA - #AACCTGTG     8880     - CTCTCCCTTC TGAGTCTTTG CTGGACGACG GGCAGGGGGT GGAGAAGTGG TG - #GGGAGGGA     8940     - GTCTGGCTCA GAGGATGACA GCGGGGCTGG GATCCAGGGC GTCTGCATCA CA - #GTCTTGTG     9000     - ACAACTGGGG GCCCACACAC ATCACTGCGG CTCTTTGAAA CTTTCAGGAA CC - #AGGGAGGG     9060     - ACTCGGCAGA GACATCTGCC AGTTCACTTG GAGTGTTCAG TCAACACCCA AA - #CTCGACAA     9120     - AGGACAGAAA GTGGAAAATG GCTGTCTCTT AGTCTAATAA ATATTGATAT GA - #AACTCAAG     9180     - TTGCTCATGG ATCAATATGC CTTTATGATC CAGCCAGCCA CTACTGTCGT AT - #CAACTCAT     9240     - GTACCCAAAC GCACTGATCT GTCTGGCTAA TGATGAGAGA TTCCCAGTAG AG - #AGCTGGCA     9300     - AGAGGTCACA GTGAGAACTG TCTGCACACA CAGCAGAGTC CACCAGTCAT CC - #TAAGGAGA     9360     - TCAGTCCTGG TGTTCATTGG AGGACTGATG TTGAAGCTGA AACTCCAATG CT - #TTGGCCAC     9420     - CTGATGTGAA GAGCTGACTC ATTTGAAAAG ACCCTGATGC TGGGAAAGAT TG - #AGGGCAGG     9480     - AGGAGAAGGG GACGACAGAG GATGAGATGG TTGGATGGCA TCACCAACAC AA - #TGGACATG     9540     - GGTTTGGGTG GACTCCAGGA GTTGGTGATG GACAGGGAGG CCTGGCGTGC TA - #CGGAAGCG     9600     - GTTTATGGGG TCACAAAGAC TGAGTGACTG AACTGAGCTG AACTGAATGG AA - #ATGAGGTA     9660     - TACAGCAAAG TGGGGATTTT TTAGATAATA AGAATATACA CATAACATAG TG - #TATACTCA     9720     - TATTTTTATG CATACCTGAA TGCTCAGTCA CTCAGTCGTA TCTGACTCTG TG - #ACCTATGG     9780     - ACCGTAGCCT TCCAGGTTTC TTCTGTCCAC AGAATTCTCC AAGGCAAGAA TA - #CTGGAGTG     9840     - GGTAGCCATT TCCTCCTCCA GGGGATCCTC CCGACCCAGG GATTGAACCG GC - #ATCTCCTG     9900     - TATTGGCAGG TGGATTCTTT ACCACTGTGC CACCAGGGAA GCCCGTGTTA CT - #CTCTATGT     9960     - CCCACTTAAT TACCAAAGCT GCTCCAAGAA AAAGCCCCTG TGCCCTCTGA GC - #TTCCCGGC     10020     - CTGCAGAGGG TGGTGGGGGT AGACTGTGAC CTGGGAACAC CCTCCCGCTT CA - #GGACTCCC     10080     - GGGCCACGTG ACCCACAGTC CTGCAGACAG CCGGGTAGCT CTGCTCTTCA AG - #GCTCATTA     10140     - TCTTTAAAAA AAACTGAGGT CTATTTTGTG ACTTCGCTGC CGTAACTTCT GA - #ACATCCAG     10200     - TGCGATGGAC AGGACCTCCT CCCCAGGCCT CAGGGGCTTC AGGGAGCCAG CC - #TTCACCTA     10260     - TGAGTCACCA GACACTCGGG GGTGGCCCCG CCTTCAGGGT GCTCACAGTC TT - #CCCATCGT     10320     - CCTGATCAAA GAGCAAGACC AATGACTTCT TAGGAGCAAG CAGACACCCA CA - #GGACACTG     10380     - AGGTTCACCA GAGCTGAGCT GTCCTTTTGA ACCTAAAGAC ACACAGCTCT CG - #AAGGTTTT     10440     - CTCTTTAATC TGGATTTAAG GCCTACTTGC CCCTCAAGAG GGAAGACAGT CC - #TGCATGTC     10500     - CCCAGGACAG CCACTCGGTG GCATCCGAGG CCACTTAGTA TTATCTGACC GC - #ACCCTGGA     10560     - ATTAATCGGT CCAAACTGGA CAAAAACCTT GGTGGGAAGT TTCATCCCAG AG - #GCCTCAAC     10620     - CATCCTGCTT TGACCACCCT GCATCTTTTT TTCTTTTATG TGTATGCATG TA - #TATATATA     10680     - TATATATTTT TTTTTTTTTC ATTTTTTGGC TGTGCTGGCT GTTCGTTGCA GT - #TCGGTGCG     10740     - CAGGCTTCTC TCTAGTTTCT CTCTAGTCTT CTCTTATCAC AGAGCAGTCT CT - #AGACGATC     10800     #       10807     - (2) INFORMATION FOR SEQ ID NO:6:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 47 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:     #                47TCGA CGATATACTC TAGACGATCG ACGCGTA     - (2) INFORMATION FOR SEQ ID NO:7:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 47 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:     #                47GTCT AGAGTATATC GTCGACGCGT CGATCGG     - (2) INFORMATION FOR SEQ ID NO:8:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 24 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:     #                24GCCT CTGG     - (2) INFORMATION FOR SEQ ID NO:9:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 24 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:     #                24TGAG CCAG     - (2) INFORMATION FOR SEQ ID NO:10:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 10 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -    (vii) IMMEDIATE SOURCE:               (B) CLONE: ZC6839     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:     #        10     - (2) INFORMATION FOR SEQ ID NO:11:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 25 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -    (vii) IMMEDIATE SOURCE:               (B) CLONE: ZC962     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:     #               25 AACG AGACA     - (2) INFORMATION FOR SEQ ID NO:12:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 45 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -    (vii) IMMEDIATE SOURCE:               (B) CLONE: ZC6303     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:     #45                AGCC ATGTGGCAGC TCACAAGCCT CCTGC     - (2) INFORMATION FOR SEQ ID NO:13:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 45 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -    (vii) IMMEDIATE SOURCE:               (B) CLONE: ZC6337     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:     #45                CGCT TGCGCCGTTG CAGCACCTGG TGGGC     - (2) INFORMATION FOR SEQ ID NO:14:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 25 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -    (vii) IMMEDIATE SOURCE:               (B) CLONE: ZC6306     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:     #               25 GTTT CTTGC     - (2) INFORMATION FOR SEQ ID NO:15:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 28 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -    (vii) IMMEDIATE SOURCE:               (B) CLONE: ZC6338     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:     #             28   CCAA CTCCTTCC     - (2) INFORMATION FOR SEQ ID NO:16:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 28 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -    (vii) IMMEDIATE SOURCE:               (B) CLONE: ZC6373     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:     #             28   TAAA AATTTAAC     - (2) INFORMATION FOR SEQ ID NO:17:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 32 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -    (vii) IMMEDIATE SOURCE:               (B) CLONE: ZC6305     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:     #          32      TTAA GTGACTGCGC TT     - (2) INFORMATION FOR SEQ ID NO:18:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 49 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -    (vii) IMMEDIATE SOURCE:               (B) CLONE: ZC6302     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:     #               49GACAC AGAAGACCAA GAAGACCAAG TAGATCCGC     - (2) INFORMATION FOR SEQ ID NO:19:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 43 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -    (vii) IMMEDIATE SOURCE:               (B) CLONE: ZC6304     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:     # 43               CTTG GTCTTCTGTG TCTCGTTTTC TTC     - (2) INFORMATION FOR SEQ ID NO:20:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 4 amino               (B) TYPE: amino acid               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:     - Arg Arg Lys Arg     - (2) INFORMATION FOR SEQ ID NO:21:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 4 amino               (B) TYPE: amino acid               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:     - Lys Arg Lys Arg     1     - (2) INFORMATION FOR SEQ ID NO:22:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 8 amino               (B) TYPE: amino acid               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:     - Ser His Leu Arg Arg Lys Arg Asp     1               5     - (2) INFORMATION FOR SEQ ID NO:23:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 6763 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: double               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:     - ACGCGTCGAC CTGCAGGTCA ACGGATCTCT GTGTCTGTTT TCATGTTAGT AC - #CACACTGT       60     - TTTGGTGGCT GTAGCTTTCA GCTACAGTCT GAAGTCATAA AGCCTGGTAC CT - #CCAGCTCT      120     - GTTCTCTCTC AAGATTGTGT TCTGCTGTTT GGGTCTTTAG TGTCTCCACA CA - #ATTTTTAG      180     - AATTGTTTGT TCTAGTTCTG TGAAAAATGA TGCTGGTATT TTGATAAGGA TT - #GCATTGAA      240     - TCTGTAAAGC TACAGATATA GTCATTGGGT AGTACAGTCA CTTTAACAAT AT - #TAACTCTT      300     - CACATCTGTG AGCATGATAT ATTTTCCCCC TCTATATCAT CTTCAATTCC TC - #CTATCAGT      360     - TTCTTTCATT GCAGTTTTCT GAGTACAGGT CTTACACCTC CTTGGTTAGA GT - #CATTCCTC      420     - AGTATTTTAT TCCTTTGATA CAATTGTGAA TGAGGTAATT TTCTTAGTTT CT - #CTTTCTGA      480     - TAGCTCATTG TTAGTGTATA TATAGAAAAG CAACAGATTT CTATGTATTA AT - #TTTGTATC      540     - CTGCAACAGA TTTCTATGTA TTAATTTTGT ATCCTGCTAC TTTACGGAAT TC - #ACTTATTA      600     - GCTTTTTGGT GACATCTTGA GGATTTTCTG AAGAAAATGG CATGGTATGG TA - #GGACAAGG      660     - TGTCATGTCA TCTGCAAACA GTGGCAGTTT TCCTTCTTCC CTTCCAACCT GG - #ATTTCTTT      720     - GATTTCTTTC TGTCTGAGTA CGACTAGGAT TCCCAATACT ATACCGAATA AA - #AGTGGCAA      780     - GAGTGGACAT CCTTGTCTTA TTTTTCTGAC CTTAGAGGAA ATGCTTTCAG TT - #TTTCACCA      840     - TTAATTATAA TGTTTACTGT GGGCTTGTCA TATGTGGCCT TCATTATATG GA - #GGTCTATT      900     - CCCTCTATAC CCACCTTGTT GAGAGTTTTT ATCATAAAAG TATGTTGAAT TT - #TGTCAAAA      960     - GTTTTTCCTG CATCTATTGA GATGATTTTT ACTCTTCAAT TCATTAATGA TT - #TTTATTCT     1020     - TCATTTTGTT AATGATTTCC ATTCTTCAAT TTGTTAACGT GGTATATCAC AT - #TGATTGAT     1080     - TTGTGGATAC CTTTGTATCC CTGGGATAAA CCTCACTTGA TCATGAGCTT TC - #AATGTATT     1140     - TTTGAATTCA CTTTGCTAAT ATTCTGTTGG GTATTTTTGC ATCTCTATTC AT - #CAATGATA     1200     - TTGGCCTAAG AAAGGTTTTG TCTGGTTTTA GTATCAGGGT GATGCTGGCC TC - #ATAGAGAG     1260     - AGTTTAGAAG CATTTCCTCC TCTTTGATTT TTCGGAATAG TTTGAGTAGG AT - #AGGTATTA     1320     - ACTCTTCTTT AAATGTTTGG GGACTTCCCT GGTGAGCCGG TGGTTGAGAA TC - #CGCCTCAG     1380     - GGATGTGGGT TTGATCCCTG GTCAGGGAAC CATTAATAAG ATCCCACATG CT - #GCAGGCAA     1440     - CAAGCCCCCA AGCTGCAACC ACTGAGCTGC AACCGCTGCA GTGCCCACAG GC - #CACGACCA     1500     - GAGAAAGCCC ACATACAGCA GGGAAGACCC AGCACAACCG GAAAAAGGAG TT - #TGGTGGAA     1560     - TACAGCTGTG AAGCCGTCTG GTCCTGGACT CCTGCTTGAG GGAATTTTTT AA - #AAATTATT     1620     - GATTCAATTT CATTACTGGT AACTGGTCTG TTCATATTTT CTATTTCTTC CG - #GGTTCAGT     1680     - CTTGGGAGAT TGTACATGCC TAGGAATGTG TCCGTTTCTT CTAGGTTGTC CA - #TTTTATTG     1740     - GACATGCATG GGAGCACACA GCACCGACCA GCGAGACTCA TGCTGGCTTC CT - #GGGGCCAG     1800     - GCTGGGGCCC CAAGCAGCAT GGCATCCTAG AGTGTGTGAA AGCCCACTGA CC - #CTGCCCAG     1860     - CCCCACAATT TCATTCTGAG AAGTGATTCC TTGCTTCTGC ACTTACAGGC CC - #AGGATCTG     1920     - ACCTGCTTCT GAGGAGCAGG GGTTTTGGCA GGACGGGGAG ATGCTGAGAG CC - #GACGGGGG     1980     - TCCAGGTCCC CTCCCAGGCC CCCCTGTCTG GGGCAGCCCT TGGGAAAGAT TG - #CCCCAGTC     2040     - TCCCTCCTAC AGTGGTCAGT CCCAGCTGCC CCAGGCCAGA GCTGCTTTAT TT - #CCGTCTCT     2100     - CTCTCTGGAT GGTATTCTCT GGAAGCTGAA GGTTCCTGAA GTTATGAATA GC - #TTTGCCCT     2160     - GAAGGGCATG GTTTGTGGTC ACGGTTCACA GGAACTTGGG AGACCCTGCA GC - #TCAGACGT     2220     - CCCGAGATTG GTGGCACCCA GATTTCCTAA GCTCGCTGGG GAACAGGGCG CT - #TGTTTCTC     2280     - CCTGGCTGAC CTCCCTCCTC CCTGCATCAC CCAGTTCTGA AAGCAGAGCG GT - #GCTGGGGT     2340     - CACAGCCTCT CGCATCTAAC GCCGGTGTCC AAACCACCCG TGCTGGTGTT CG - #GGGGGCTA     2400     - CCTATGGGGA AGGGCTTCTC ACTGCAGTGG TGCCCCCCGT CCCCTCTGAG AT - #CAGAAGTC     2460     - CCAGTCCGGA CGTCAAACAG GCCGAGCTCC CTCCAGAGGC TCCAGGGAGG GA - #TCCTTGCC     2520     - CCCCCGCTGC TGCCTCCAGC TCCTGGTGCC GCACCCTTGA GCCTGATCTT GT - #AGACGCCT     2580     - CAGTCTAGTC TCTGCCTCCG TGTTCACACG CCTTCTCCCC ATGTCCCCTC CG - #TGTCCCCG     2640     - TTTTCTCTCA CAAGGACACC GGACATTAGA TTAGCCCCTG TTCCAGCCTC AC - #CTGAACAG     2700     - CTCACATCTG TAAAGACCTA GATTCCAAAC AAGATTCCAA CCTGAAGTTC CC - #GGTGGATG     2760     - TGAGTTCTGG GGCGACATCC TTCAACCCCA TCACAGCTTG CAGTTCATCG CA - #AAACATGG     2820     - AACCTGGGGT TTATCGTAAA ACCCAGGTTC TTCATGAAAC ACTGAGCTTC GA - #GGCTTGTT     2880     - GCAAGAATTA AAGGTGCTAA TACAGATCAG GGCAAGGACT GAAGCTGGCT AA - #GCCTCCTC     2940     - TTTCCATCAC AGGAAAGGGG GGCCTGGGGG CGGCTGGAGG TCTGCTCCCG TG - #AGTGAGCT     3000     - CTTTCCTGCT ACAGTCACCA ACAGTCTCTC TGGGAAGGAA ACCAGAGGCC AG - #AGAGCAAG     3060     - CCGGAGCTAG TTTAGGAGAC CCCTGAACCT CCACCCAAGA TGCTGACCAG CC - #AGCGGGCC     3120     - CCCTGGAAAG ACCCTACAGT TCAGGGGGGA AGAGGGGCTG ACCCGCCAGG TC - #CCTGCTAT     3180     - CAGGAGACAT CCCCGCTATC AGGAGATTCC CCCACCTTGC TCCCGTTCCC CT - #ATCCCAAT     3240     - ACGCCCACCC CACCCCTGTG ATGAGCAGTT TAGTCACTTA GAATGTCAAC TG - #AAGGCTTT     3300     - TGCATCCCCT TTGCCAGAGG CACAAGGCAC CCACAGCCTG CTGGGTACCG AC - #GCCCATGT     3360     - GGATTCAGCC AGGAGGCCTG TCCTGCACCC TCCCTGCTCG GGCCCCCTCT GT - #GCTCAGCA     3420     - ACACACCCAG CACCAGCATT CCCGCTGCTC CTGAGGTCTG CAGGCAGCTC GC - #TGTAGCCT     3480     - GAGCGGTGTG GAGGGAAGTG TCCTGGGAGA TTTAAAATGT GAGAGGCGGG AG - #GTGGGAGG     3540     - TTGGGCCCTG TGGGCCTGCC CATCCCACGT GCCTGCATTA GCCCCAGTGC TG - #CTCAGCCG     3600     - TGCCCCCGCC GCAGGGGTCA GGTCACTTTC CCGTCCTGGG GTTATTATGA CT - #CTTGTCAT     3660     - TGCCATTGCC ATTTTTGCTA CCCTAACTGG GCAGCAGGTG CTTGCAGAGC CC - #TCGATACC     3720     - GACCAGGTCC TCCCTCGGAG CTCGACCTGA ACCCCATGTC ACCCTTGCCC CA - #GCCTGCAG     3780     - AGGGTGGGTG ACTGCAGAGA TCCCTTCACC CAAGGCCACG GTCACATGGT TT - #GGAGGAGC     3840     - TGGTGCCCAA GGCAGAGGCC ACCCTCCAGG ACACACCTGT CCCCAGTGCT GG - #CTCTGACC     3900     - TGTCCTTGTC TAAGAGGCTG ACCCCGGAAG TGTTCCTGGC ACTGGCAGCC AG - #CCTGGACC     3960     - CAGAGTCCAG ACACCCACCT GTGCCCCCGC TTCTGGGGTC TACCAGGAAC CG - #TCTAGGCC     4020     - CAGAGGGGAC TTCCTGCTTG GCCTTGGATG GAAGAAGGCC TCCTATTGTC CT - #CGTAGAGG     4080     - AAGCCACCCC GGGGCCTGAG GATGAGCCAA GTGGGATTCC GGGAACCGCG TG - #GCTGGGGG     4140     - CCCAGCCCGG GCTGGCTGGC CTGCATGCCT CCTGTATAAG GCCCCAAGCC TG - #CTGTCTCA     4200     - GCCCTCCACT CCCTGCAGAG CTCAGAAGCA CGACCCCAGG GATATCATCG AT - #AAGCTTGG     4260     - ATCCCCTGCC GGTGCCTCTG GGGTAAGCTG CCTGCCCTGC CCCACGTCCT GG - #GCACACAC     4320     - ATGGGGTAGG GGGTCTTGGT GGGGCCTGGG ACCCCACATC AGGCCCTGGG GT - #CCCCCCTG     4380     - TGAGAATGGC TGGAAGCTGG GGTCCCTCCT GGCGACTGCA GAGCTGGCTG GC - #CGCGTGCC     4440     - ACTCTTGTGG GTGACCTGTG TCCTGGCCTC ACACACTGAC CTCCTCCAGC TC - #CTTCCAGC     4500     - AGAGCTAAGG CTAAGTGAGC CAGAATGGTA CCTAAGGGGA GGCTAGCGGT CC - #TTCTCCCG     4560     - AGGAGGGGCT GTCCTGGAAC CACCAGCCAT GGAGAGGCTG GCAAGGGTCT GG - #CAGGTGCC     4620     - CCAGGAATCA CAGGGGGGCC CCATGTCCAT TTCAGGGCCC GGGAGCCTTG GA - #CTCCTCTG     4680     - GGGACAGACG ACGTCACCAC CGCCCCCCCC CCATCAGGGG GACTAGAAGG GA - #CCAGGACT     4740     - GCAGTCACCC TTCCTGGGAC CCAGGCCCCT CCAGGCCCCT CCTGGGGCTC CT - #GCTCTGGG     4800     - CAGCTTCTCC TTCACCAATA AAGGCATAAA CCTGTGCTCT CCCTTCTGAG TC - #TTTGCTGG     4860     - ACGACGGGCA GGGGGTGGAG AAGTGGTGGG GAGGGAGTCT GGCTCAGAGG AT - #GACAGCGG     4920     - GGCTGGGATC CAGGGCGTCT GCATCACAGT CTTGTGACAA CTGGGGGCCC AC - #ACACATCA     4980     - CTGCGGCTCT TTGAAACTTT CAGGAACCAG GGAGGGACTC GGCAGAGACA TC - #TGCCAGTT     5040     - CACTTGGAGT GTTCAGTCAA CACCCAAACT CGACAAAGGA CAGAAAGTGG AA - #AATGGCTG     5100     - TCTCTTAGTC TAATAAATAT TGATATGAAA CTCAAGTTGC TCATGGATCA AT - #ATGCCTTT     5160     - ATGATCCAGC CAGCCACTAC TGTCGTATCA ACTCATGTAC CCAAACGCAC TG - #ATCTGTCT     5220     - GGCTAATGAT GAGAGATTCC CAGTAGAGAG CTGGCAAGAG GTCACAGTGA GA - #ACTGTCTG     5280     - CACACACAGC AGAGTCCACC AGTCATCCTA AGGAGATCAG TCCTGGTGTT CA - #TTGGAGGA     5340     - CTGATGTTGA AGCTGAAACT CCAATGCTTT GGCCACCTGA TGTGAAGAGC TG - #ACTCATTT     5400     - GAAAAGACCC TGATGCTGGG AAAGATTGAG GGCAGGAGGA GAAGGGGACG AC - #AGAGGATG     5460     - AGATGGTTGG ATGGCATCAC CAACACAATG GACATGGGTT TGGGTGGACT CC - #AGGAGTTG     5520     - GTGATGGACA GGGAGGCCTG GCGTGCTACG GAAGCGGTTT ATGGGGTCAC AA - #AGACTGAG     5580     - TGACTGAACT GAGCTGAACT GAATGGAAAT GAGGTATACA GCAAAGTGGG GA - #TTTTTTAG     5640     - ATAATAAGAA TATACACATA ACATAGTGTA TACTCATATT TTTATGCATA CC - #TGAATGCT     5700     - CAGTCACTCA GTCGTATCTG ACTCTGTGAC CTATGGACCG TAGCCTTCCA GG - #TTTCTTCT     5760     - GTCCACAGAA TTCTCCAAGG CAAGAATACT GGAGTGGGTA GCCATTTCCT CC - #TCCAGGGG     5820     - ATCCTCCCGA CCCAGGGATT GAACCGGCAT CTCCTGTATT GGCAGGTGGA TT - #CTTTACCA     5880     - CTGTGCCACC AGGGAAGCCC GTGTTACTCT CTATGTCCCA CTTAATTACC AA - #AGCTGCTC     5940     - CAAGAAAAAG CCCCTGTGCC CTCTGAGCTT CCCGGCCTGC AGAGGGTGGT GG - #GGGTAGAC     6000     - TGTGACCTGG GAACACCCTC CCGCTTCAGG ACTCCCGGGC CACGTGACCC AC - #AGTCCTGC     6060     - AGACAGCCGG GTAGCTCTGC TCTTCAAGGC TCATTATCTT TAAAAAAAAC TG - #AGGTCTAT     6120     - TTTGTGACTT CGCTGCCGTA ACTTCTGAAC ATCCAGTGCG ATGGACAGGA CC - #TCCTCCCC     6180     - AGGCCTCAGG GGCTTCAGGG AGCCAGCCTT CACCTATGAG TCACCAGACA CT - #CGGGGGTG     6240     - GCCCCGCCTT CAGGGTGCTC ACAGTCTTCC CATCGTCCTG ATCAAAGAGC AA - #GACCAATG     6300     - ACTTCTTAGG AGCAAGCAGA CACCCACAGG ACACTGAGGT TCACCAGAGC TG - #AGCTGTCC     6360     - TTTTGAACCT AAAGACACAC AGCTCTCGAA GGTTTTCTCT TTAATCTGGA TT - #TAAGGCCT     6420     - ACTTGCCCCT CAAGAGGGAA GACAGTCCTG CATGTCCCCA GGACAGCCAC TC - #GGTGGCAT     6480     - CCGAGGCCAC TTAGTATTAT CTGACCGCAC CCTGGAATTA ATCGGTCCAA AC - #TGGACAAA     6540     - AACCTTGGTG GGAAGTTTCA TCCCAGAGGC CTCAACCATC CTGCTTTGAC CA - #CCCTGCAT     6600     - CTTTTTTTCT TTTATGTGTA TGCATGTATA TATATATATA TATTTTTTTT TT - #TTTCATTT     6660     - TTTGGCTGTG CTGGCTGTTC GTTGCAGTTC GGTGCGCAGG CTTCTCTCTA GT - #TTCTCTCT     6720     #                 676 - #3GTCTCTAG ACGATCGACG CGT     - (2) INFORMATION FOR SEQ ID NO:24:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 5 amino               (B) TYPE: amino acid               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:     - Arg Ile Arg Lys Arg     1               5     - (2) INFORMATION FOR SEQ ID NO:25:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 5 amino               (B) TYPE: amino acid               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:     - Gln Arg Arg Lys Arg     1               5     __________________________________________________________________________ 

We claim:
 1. A method for producing protein C in a transgenic non-human mammal comprising:providing a DNA construct comprising a first DNA segment encoding a secretion signal and a protein C propeptide operably linked to a second DNA segment encoding protein C, wherein the encoded protein C comprises a two-chain cleavage site modified from Lysine (Lys)-Arginine (Arg) to R₁ -R₂ -R₃ -R₄, and wherein each of R₁, R₂, R₃, R₄ is individually Lys or Arg, and wherein said first and second segments are operably linked to additional DNA segments required for expression of the protein C DNA in a mammary gland of a host female non-human mammal; introducing said DNA construct into a fertilized egg of a non-human mammalian species; inserting said egg into an oviduct or uterus of a female of said species to obtain offspring carrying said DNA construct; breeding said offspring to produce female progeny that express said first and second DNA segments and produce milk containing protein C encoded by said second segment, wherein said protein has anticoagulant activity upon activation; collecting milk from said female progeny; and recovering the protein C from the milk.
 2. The method of claim 1, further comprising the step of activating the protein C.
 3. The method of claim 1, wherein R₁ -R₂ -R₃ -R₄ is Arg-Arg-Lys-Arg (SEQ ID NO: 20).
 4. The method of claim 1, wherein said species is selected from sheep, rabbits, cattle and goats.
 5. The method of claim 1, wherein each of said first and second DNA segments comprises an intron.
 6. The method of claim 1, wherein the second DNA segment comprises a DNA sequence of nucleotides as shown in Seq. ID NO: 1 or Seq. ID. NO:
 3. 7. The method of claim 6, wherein the second DNA segment comprises the DNA sequence of nucleotides as shown in SEQ. ID. NO:
 1. 8. The method of claim 1, wherein the additional DNA segments comprise a transcriptional promoter selected from the group consisting of casein, β-lactoglobulin, α-lactalbumin and whey acidic protein gene promoters.
 9. The method of claim 8, wherein the transcriptional promoter is the β-lactoglobulin gene promoter.
 10. A transgenic non-human female mammal whose genome comprises a DNA construct comprising a first EDNA segment encoding a secretion signal and a protein C propeptide operably linked to a second DNA segment encoding protein C, wherein the encoded protein C comprises a two-chain cleavage site modified from Lysine (Lys)-Arginine (Arg) to R₁ -R₂ -R₃ -R₄, and wherein each of R₁, R₂ , R₃, R₄ is individually Lys or Arg, and wherein said first and second segments are operably linked to additional DNA segments required for the expression of the protein C DNA in the mammary gland of the mammal such that expression of the DNA construct results in the production of recoverable amounts of human protein C in milk of the mammal wherein at least 90% of the human protein C in the milk is two chain protein C.
 11. A process for producing a transgenic offspring of a non-human mammal comprising:providing a DNA construct comprising a first DNA segment encoding a secretion signal and a protein C propeptide operably linked to a second DNA segment encoding protein C, wherein the encoded protein C comprises a two-chain cleavage site modified from Lys-Arg to R₁ -R₂ -R₃ -R₄, and wherein each of R₁, R₂, R₃, R₄, is individually Lys or Arg, and wherein said first and second segments are operably linked to additional DNA segments required for expression of the protein C DNA in the mammary gland of a host female mammal; introducing said DNA construct into a fertilized egg of a non-human mammalian species; and inserting said egg into an oviduct or uterus of a female of said species to obtain offspring carrying said DNA construct.
 12. The process according to claim 11, wherein R₁ -R₂ -R₃ -R₄ is Arg-Arg-Lys-Arg (SEQ ID NO: 20).
 13. The process according to claim 11, wherein the offspring is female.
 14. The process according to claim 11, wherein the offspring is male.
 15. A non-human mammal produced according to the process of claim 11, wherein expression of the DNA construct results in the production of recoverable amounts of human protein C in milk, wherein the protein C has anticoagulant activity upon activation.
 16. A non-human mammal of claim 15, wherein the mammal is female.
 17. A non-human mammalian embryo whose genome comprises a DNA construct comprising a first DNA segment encoding a secretion signal and a protein C propeptide operably linked to a second DNA segment encoding protein C, wherein the encoded protein C comprises a two-chain cleavage site modified from Lysine (Lys)-Arginine (Arg) to R₁ -R₂ -R₃ -R₄, and wherein each of R₁, R₂, R₃, R₄ is individually Lys or Arg, and wherein said first and second segments are operably linked to additional DNA segments required for the expression of the protein C DNA in the mammary gland of the mammal such that the embryo develops into a transgenic non-human mammal such that expression of the DNA construct in the transgenic non-human mammal results in the production of recoverable amounts of human protein C in milk of the mammal wherein at least 90% of the human protein C in the milk is two chain protein C. 